Proteases

ABSTRACT

The present invention relates to proteases of high specific activity homologous to proteases derived from  Nocardiopsis , and the production thereof by the wild-type, and in recombinant host cells including transgenic plants and non-human transgenic animals. The proteases are effective in animal feed, and detergents. Characteristic structural features of relevance for the specific activity of these proteases of peptidase family S2A or S1E are disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority or the benefit under 35 U.S.C. 119 ofDanish application nos. PA 2003 00913 filed Jun. 19, 2003, PA 2003 01492filed Oct. 10, 2003, and PA 2003 00332 filed Mar. 1, 2004, and U.S.provisional application Nos. 60/480,024 filed Jun. 20, 2003, 60/510,411filed Oct. 10, 2003, and 60/549,349 filed Mar. 2, 2004, the contents ofwhich are fully incorporated herein by reference.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to an isolated polypeptide having proteaseactivity and being homologous to Nocardiopsis proteases, as well asisolated nucleic acid sequences encoding it. The invention furthermorerelates to nucleic acid constructs, vectors, and host cells, includingtransgenic plants and non-human animals, comprising these nucleic acidsequences, as well as methods for producing and using the protease, inparticular within animal feed.

The protease of the invention has a high specific activity.Characteristic structural features of relevance for the high specificactivity of proteases of peptidase family S2A or S1E are disclosed.

Proteases derived from Nocardiopsis sp. NRRL 18262 and Nocardiopsisdassonvillei NRRL 18133 are disclosed in WO 88/03947. The DNA and aminoacid sequences of the protease derived from Nocardiopsis sp. NRRL 18262are shown in DK application no. 1996 00013. WO 01/58276 discloses theuse in animal feed of acid-stable proteases related to the proteasederived from Nocardiopsis sp. NRRL 18262, as well as a protease derivedfrom Nocardiopsis alba DSM 14010. These proteases, however, have a lowspecific activity.

JP 2-255081-A discloses a protease derived from Nocardiopsis sp. strainOPC-210 (FERM P-10508), however without sequence information. The strainis no longer available, as the deposit was withdrawn.

DD 200432|8 discloses a proteolytic preparation derived fromNocardiopsis dassonvillei strain ZIMET 43647, however without sequenceinformation. The strain appears to be no longer available.

JP 2003284571-A, published after the first filing date of the presentinvention, discloses the amino acid sequence and the corresponding DNAsequence of a protease derived from Nocardiopsis sp. TOA-1 (FERMP-18676). The sequence has been entered in GENESEQP with no. ADF43564.

The most homologous prior art protease which is not a Nocardiopsisprotease is SapII_Streptomyces_sp_sptrembl_q55353, the mature part ofwhich has an amino acid identity of 61.5%, and 63.5%, respectively, tothe mature parts of SEQ ID NOs: 2 and 6, respectively. The correspondingDNA identities are 70.3%, and 72.7%, to SEQ ID NOs: 1 and 5,respectively. The mature part of a related Streptomyces protease, viz.SapII_Streptomyces_sp_sptrembl_q55352 has a slightly higher percentageidentity to the mature part of SEQ ID NO: 1, viz. 70.8%.

It is an object of the present invention to provide proteases of a highspecific activity homologous to Nocardiopsis proteases, in particularwith a potential for use in animal feed and/or detergents.

SUMMARY OF THE INVENTION

Proteases of high specific activity were isolated and characterized,viz. a protease derived from Nocardiopsis alba DSM 15647 (see SEQ IDNOs: 1 and 2), and a protease derived from Nocardiopsis dassonvilleisubsp. dassonvillei DSM 43235 (see SEQ ID NOs: 5 and 6).

In a first aspect, the invention relates to an isolated polypeptidehaving protease activity, and having a specific activity on haemoglobinat pH 7.5 and 25° C. of at least 39 AU/g, wherein the polypeptide isselected from the group consisting of: (a) a polypeptide having an aminoacid sequence which has a degree of identity to amino acids 1 to 188 ofSEQ ID NO: 2, and/or to amino acids 1–192 of SEQ ID NO: 6, of at least65%; (b) a polypeptide which is encoded by a nucleic acid sequence whichhybridizes under low stringency conditions with nucleotides 502–1065 ofSEQ ID NO: 1, and/or nucleotides 568–1143 of SEQ ID NO: 5; (c) apolypeptide which is encoded by a nucleic acid sequence which has adegree of identity to nucleotides 502–1065 of SEQ ID NO: 1, and/ornucleotides 568–1143 of SEQ ID NO: 5, of at least 74%. The inventionalso relates to isolated nucleic acid sequences encoding such proteases;nucleic acid constructs, vectors, and host cells comprising the nucleicacid sequences; as well as methods for producing and using theproteases, in particular within animal feed.

In a second aspect, the invention relates to:

-   A. An isolated polypeptide of peptidase family S2A and/or peptidase    family S1E having protease activity and having an amino sequence    comprising at least one of the following amino acids at the position    indicated: 25S, 38T, 42P, 44S, 49Q, 54R, 62S, 89S, 91S, 92S, 95A,    99Q,100I, 114V, 120T, 125Q, 129Q, 131L, 135N, 147F, 151S, 165S,    166F, 171Y, 176N, 179L, 180S, 184L, and/or 185T; preferably 25S,    38T, 42P, 44S, 54R, 62S, 125Q, 131L, 165S, 171Y, 176N, 179L, 180S,    184L, and/or 185T; more preferably together with at least one of    24A, 51V, 53E, 86A, 87T, 96I, and/or 186L; and/or together with    (H35+D61+S143); wherein each position corresponds to a position of    SEQ ID NO: 2.-   B. The polypeptide of A which comprises at least one of the    following amino acids at the position indicated: 38T, 92S, 120T,    125Q, 131L, 135N, 147F, 151S, 165S, and/or 171Y.-   C. The polypeptide of A which comprises at least one of the    following amino acids at the position indicated: 25S, 42P, 44S, 49Q,    54R, 62S, 89S, 91S, 95A, 99Q, 100I, 114V, 129Q, 166F, 176N, 179L,    180S, 184L, and/or 185T.-   D. The polypeptide of any one of A, B, or C, which has a Tm of at    least 78° C. as measured by DSC in 10 mM sodium phosphate, 50 mM    sodium chloride, pH 7.0; a relative activity at pH9 and 80° C. of at    least 0.40; and/or a specific activity on haemoglobin at pH 7.5 and    25° C. of at least 39 AU/g.-   E. The polypeptide of any one of A, B, C, or D, which has a    percentage of identity to amino acids −167 to 188, preferably 1 to    188, of SEQ ID NO: 2, and/or to amino acids −160 to 192, preferably    1–192, of SEQ ID NO: 6 of at least 65%, more preferably a percentage    of identity to amino acids 1–188, or 167 to 188, of SEQ ID NO: 2 of    at least 50%.-   F. The polypeptide of any one of A, B, C, D, or E, which is i) a    bacterial protease; ii) a protease of the phylum    Actinobacteria; iii) of the class Actinobacteria; iv) of the order    Actinomycetales v) of the family Nocardiopsaceae; vi) of the genus    Nocardiopsis; and/or a protease derived from vii) Nocardiopsis    species such as Nocardiopsis alba, Nocardiopsis antarctica,    Nocardiopsis prasina, Nocardiopsis composta, Nocardiopsis exhalans,    Nocardiopsis halophila, Nocardiopsis halotolerans, Nocardiopsis    kunsanensis, Nocardiopsis listeri, Nocardiopsis lucentensis,    Nocardiopsis metallicus, Nocardiopsis synnemataformans, Nocardiopsis    trehalosi, Nocardiopsis tropica, Nocardiopsis umidischolae,    Nocardiopsis xinjiangensis, or Nocardiopsis dassonvillei; and,    optionally, also from Nocardiopsis alkaliphila, e.g. a protease    derived from Nocardiopsis alba, for example Nocardiopsis alba DSM    15647, or a protease derived from Nocardiopsis dassonvillei, for    example Nocardiopsis dassonvillei subsp. dassonvillei DSM 43235,    such as a polypeptide with the amino acid sequence of amino acids    −167 to 188, preferably 1–188, of SEQ ID NO: 2, and/or amino acids    −160 to 192, preferably 1–192, of SEQ ID NO: 6.-   G. An isolated nucleic acid sequence comprising a nucleic acid    sequence which encodes the polypeptide of any one of A, B, C, D, E,    or F.-   H. A nucleic acid construct comprising the nucleic acid sequence of    G operably linked to one or more control sequences that direct the    production of the polypeptide in a suitable expression host.-   I. A recombinant expression vector comprising the nucleic acid    construct of H.-   J. A recombinant host cell comprising the nucleic acid construct of    H or the vector of I.-   K. A method for producing a polypeptide of any one A, B, C, D, E, or    F, the method comprising: (a) cultivating a recombinant host cell of    K to produce a supernatant comprising the polypeptide; and (b)    recovering the polypeptide.-   L. A transgenic plant, or plant part, capable of expressing the    polypeptide of any one of A, B, C, D, E,or F.-   M. A transgenic, non-human animal, or products, or elements thereof,    being capable of expressing the polypeptide of any one of A, B, C,    D, E, or F.-   N. Use of at least one polypeptide as defined in any one of A, B, C,    D, E, or F, (i) in animal feed; (ii) in the preparation of a    composition for use in animal feed; (iii) for improving the    nutritional value of an animal feed; (iv) for increasing digestible    and/or soluble protein in animal diets; (v) for increasing the    degree of hydrolysis of proteins in animal diets; and/or (vi) for    the treatment of proteins.-   O. An animal feed additive comprising at least one polypeptide as    defined in any one of A, B, C, D, E, or F; and (a) at least one    fat-soluble vitamin, and/or (b) at least one water-soluble vitamin,    and/or (c) at least one trace mineral.-   P. An animal feed composition having a crude protein content of 50    to 800 g/kg and comprising at least one polypeptide as defined in    any one of A, B, C, D, E, or F, or at least one feed additive of O.-   Q. A composition comprising at least one polypeptide as defined in    any one of A, B, C, D, E, or F, together with at least one other    enzyme selected from amongst alpha-amylase (EC 3.2.1.1), phytase (EC    3.1.3.8 or 3.1.3.26); xylanase (EC 3.2.1.8); galactanase (EC    3.2.1.89); alpha-galactosidase (EC 3.2.1.22); protease (EC 3.4.−.−),    phospholipase A1(EC 3.1.1.32); phospholipase A2 (EC 3.1.1.4);    lysophospholipase (EC 3.1.1.5); phospholipase C (3.1.4.3);    phospholipase D (EC 3.1.4.4); and/or beta-glucanase (EC 3.2.1.4 or    EC 3.2.1.6).-   R. Use of at least one polypeptide as defined in any one of A, B, C,    D, E, or F, in detergents.

In a third aspect, the invention relates to:

-   a. An isolated polypeptide having protease activity, selected from    the group consisting of: (a) a polypeptide having an amino acid    sequence which has a degree of identity to amino acids 1 to 188 of    SEQ ID NO: 2, of at least 86%, and/or to amino acids 1–192 of SEQ ID    NO: 6 of at least 72%, (b) a polypeptide which is encoded by a    nucleic acid sequence which hybridizes under medium-high stringency    conditions with (i) any one of nucleotides 502–1065 of SEQ ID NO: 1,    and/or nucleotides 568–1143 of SEQ ID NO: 5, (ii) a subsequence    of (i) of at least 100 nucleotides; and/or (iii) a complementary    strand of any one of (i)–(ii); (c) a variant of the polypeptide    having an amino acid sequence of amino acids 1 to 188 of SEQ ID NO:    2, or amino acids 1–192 of SEQ ID NO: 6, comprising a substitution,    deletion, extension, and/or insertion of one or more amino    acids; (d) an allelic variant of (a), (b), or (c); and (e) a    fragment of (a), (b), (c), or (d) that has protease activity;-   b. An isolated nucleic acid sequence comprising a nucleic acid    sequence which (a) encodes the polypeptide of a; (b) encodes a    polypeptide having protease activity, and which hybridizes under    medium-high stringency conditions with (i) any one of nucleotides    502–1065 of SEQ ID NO: 1, and/or nucleotides 568–1143 of SEQ ID NO:    5, (ii) a subsequence of (i) of at least 100 nucleotides;    and/or (ii) a complementary strand of any one of (i)–(ii);    and/or (c) encodes a polypeptide having protease activity and which    has a degree of identity (i) to nucleotides 502–1065 SEQ ID NO: 1 of    at least 86%, and/or to nucleotides 568–1143 of SEQ ID NO: 5 of at    least 82%;-   c. An isolated nucleic acid sequence produced by (a) hybridizing a    DNA under medium-high stringency conditions with (i) any one of    nucleotides 502–1065 of SEQ ID NO: 1, and/or nucleotides 568–1143 of    SEQ ID NO: 5; (ii) a subsequence of of (i) of at least 100    nucleotides; and/or (iii) a complementary strand of any one of    (i)–(ii); and (b) isolating the nucleic acid sequence;-   d. A nucleic acid construct comprising the nucleic acid sequence of    any one of b, or c, operably linked to one or more control sequences    that direct the production of the polypeptide in a suitable    expression host;-   e. A recombinant expression vector comprising the nucleic acid    construct of d;-   f. A recombinant host cell comprising the nucleic acid construct of    d or the vector of e;-   g. A method for producing a polypeptide of a, the method    comprising: (a) cultivating a recombinant host cell of f to produce    a supernatant comprising the polypeptide; and (b) recovering the    polypeptide;-   h. A transgenic plant, or plant part, capable of expressing the    polypeptide of a;-   i. A transgenic, non-human animal, or products, or elements thereof,    being capable of expressing the polypeptide of a;-   j. A method for producing a polypeptide of a, the method    comprising (a) cultivating any one of the following strains: (i)    Nocardiopsis dassonvillei subsp. dassonvillei DSM 43235, or    Nocardiopsis alba DSM 15647, and (b) recovering the polypeptide;-   k. Use of at least one polypeptide as defined in a (i) in animal    feed; (ii) in the preparation of a composition for use in animal    feed; (iii) for improving the nutritional value of an animal    feed; (iv) for increasing digestible and/or soluble protein in    animal diets; (v) for increasing the degree of hydrolysis of    proteins in animal diets; and/or (vi) for the treatment of proteins;-   l. An animal feed additive comprising at least one polypeptide as    defined in a; and (a) at least one fat-soluble vitamin, and/or (b)    at least one water-soluble vitamin, and/or (c) at least one trace    mineral;-   m. An animal feed composition having a crude protein content of 50    to 800 g/kg and comprising at least one polypeptide as defined in a,    or at least one feed additive of /;-   n. A composition comprising at least one polypeptide as defined in    a, together with at least one other enzyme selected from amongst    alpha-amylase (EC 3.2.1.1), phytase (EC 3.1.3.8 or 3.1.3.26);    xylanase (EC 3.2.1.8); galactanase (EC 3.2.1.89);    alpha-galactosidase (EC 3.2.1.22); protease (EC 3.4.−.−),    phospholipase A1 (EC 3.1.1.32); phospholipase A2 (EC 3.1.1.4);    lysophospholipase (EC 3.1.1.5); phospholipase C (3.1.4.3);    phospholipase D (EC 3.1.4.4); and/or beta-glucanase (EC 3.2.1.4 or    EC 3.2.1.6); as well as-   o. Use of at least one polypeptide as defined in a in detergents.

In a fourth aspect, the invention relates to: an an isolated polypeptidehaving protease activity, selected from the group consisting of: (a) apolypeptide having an amino acid sequence which has a degree of identityto amino acids 1 to 188 of SEQ ID NO: 2 of at least 84%; (b) apolypeptide having an amino acid sequence which has a degree of identityto amino acids −167 to 188 of SEQ ID NO: 2 of at least 78%; (c) apolypeptide which is encoded by a nucleic acid sequence which hybridizesunder medium-high stringency conditions with (i) DNA encoding a proteaseobtainable from genomic DNA from Nocardiopsis alba DSM 15647 by use ofprimers SEQ ID NOs: 3 and 4; (ii) nucleotides 502–1065 of SEQ ID NO: 1;(iii) nucleotides 1–1065 of SEQ ID NO: 1; (iv) a subsequence of (i) or(ii) or (iii) of at least 100 nucleotides; and/or (v) a complementarystrand of (i), (ii), (iii) or (iv); (d) a variant of the polypeptidehaving an amino acid sequence of amino acids 1 to 188, or −167 to 188 ofSEQ ID NO: 2, comprising a substitution, deletion, extension, and/orinsertion of one or more amino acids; (e) an allelic variant of (a), (b)or (c); and (f) a fragment of (a), (b), (c), (d) or (e) that hasprotease activity.

An isolated polypeptide having protease activity, and having a meltingtemperature (T_(m)) of at least 78° C., as determined by DifferentialScanning Calorimetry (DSC) in a 10 mM sodium phosphate, 50 mM sodiumchloride buffer, pH 7.0, using a constant scan rate of 1.5° C./min,wherein the polypeptide is selected from the group consisting of: (a) apolypeptide having an amino acid sequence which has a degree of identityto amino acids 1 to 188 of SEQ ID NO: 2 of at least 50%; (b) apolypeptide having an amino acid sequence which has a degree of identityto amino acids −167 to 188 of SEQ ID NO: 2; (c) a polypeptide which isencoded by a nucleic acid sequence which hybridizes under low stringencyconditions with (i) DNA encoding a protease obtainable from genomic DNAfrom Nocardiopsis alba DSM 15647 by use of primers SEQ ID NOs: 3 and 4;(ii) nucleotides 502–1065 of SEQ ID NO: 1; (iii) nucleotides 1–1065 ofSEQ ID NO: 1; (iv) a subsequence of (i) or (ii) or (iii) of at least 100nucleotides; and/or (v) a complementary strand of (i), (ii), (iii) or(iv); (d) a variant of the polypeptide having an amino acid sequence ofamino acids 1 to 188, or −167 to 188 of SEQ ID NO: 2, comprising asubstitution, deletion, extension, and/or insertion of one or more aminoacids; (e) an allelic variant of (a), (b) or (c); and (f) a fragment of(a), (b), (c), (d) or (e) that has protease activity.

An isolated nucleic acid sequence comprising a nucleic acid sequencewhich (a) encodes the polypeptide as defined just above; (b) encodes apolypeptide having protease activity, and which hybridizes undermedium-high stringency conditions with (i) DNA encoding a proteaseobtainable from genomic DNA from Nocardiopsis alba DSM 15647 by use ofprimers SEQ ID NOs: 3 and 4, (ii) nucleotides 502–1065 or 1–1065 of SEQID NO: 1; (iii) a subsequence of (i) or (ii) of at least 100nucleotides; and/or (iv) a complementary strand of (i), (ii), or (iii);(c) encodes a polypeptide having protease activity and which has adegree of identity to nucleotides 502–1065 SEQ ID NO: 1 of at least 86%;and/or (d) encodes a polypeptide having protease activity and which hasa degree of identity to nucleotides 1–1065 SEQ ID NO: 1 of at least 82%.

An isolated nucleic acid sequence comprising a nucleic acid sequencewhich encodes a polypeptide having protease activity and a meltingtemperature (T_(m)) of at least 78° C., as determined by DifferentialScanning Calorimetry (DSC) in a 10 mM sodium phosphate, 50 mM sodiumchloride buffer, pH 7.0, using a constant scan rate of 1.5° C./min,wherein the nucleic acid sequence (a) encodes the polypeptide with a Tmof at least 78° C. as defined above; (b) hybridizes under low stringencyconditions with (i) DNA encoding a protease obtainable from genomic DNAfrom Nocardiopsis alba DSM 15647 by use of primers SEQ ID NO's: 3 and 4;(ii) nucleotides 502–1065 or 1–1065 of SEQ ID NO: 1; (iii) a subsequenceof (i) or (ii) of at least 100 nucleotides; and/or (iv) a complementarystrand of (i), (ii), or (iii); (c) has a degree of identity tonucleotides 502–1065 of SEQ ID NO: 1 of at least 50%; and/or (d) has adegree of identity to nucleotides 1–1065 of SEQ ID NO: 1 of at least50%.

An isolated nucleic acid sequence produced by (a) hybridizing a DNAunder medium-high stringency conditions with (i) DNA encoding a proteaseobtainable from genomic DNA from Nocardiopsis alba DSM 15647 by use ofprimers SEQ ID NOs: 3 and 4; (ii) nucleotides 502–1065 or 1–1065 of SEQID NO: 1; (iii) a subsequence of (i) or (ii) of at least 100nucleotides; or (iv) a complementary strand of (i), (ii) or (iii); and(b) isolating the nucleic acid sequence.

A nucleic acid construct comprising any of the three nucleic acidsequences defined in any of the three paragraphs immediately above thepresent, operably linked to one or more control sequences that directthe production of the polypeptide in a suitable expression host.

A recombinant expression vector comprising the nucleic acid construct.

A recombinant host cell comprising the nucleic acid construct or thevector.

A method for producing a polypeptide as defined above, the methodcomprising: (a) cultivating a recombinant host cell of claim 8 toproduce a supernatant comprising the polypeptide; and (b) recovering thepolypeptide.

A transgenic plant, or plant part, capable of expressing the polypeptideas defined above.

A transgenic, non-human animal, or products, or elements thereof,capable of expressing the above-defined polypeptide.

Use of at least one of the polypeptides as defined above (i) in animalfeed; (ii) in the preparation of a composition for use in animal feed;(iii) for improving the nutritional value of an animal feed; (iv) forincreasing digestible and/or soluble protein in animal diets; (v) forincreasing the degree of hydrolysis of proteins in animal diets; and/or(vi) for the treatment of vegetable proteins.

An animal feed additive comprising at least one polypeptide as definedabove; and (a) at least one fat-soluble vitamin, and/or (b) at least onewater-soluble vitamin, and/or (c) at least one trace mineral.

An animal feed composition having a crude protein content of 50 to 800g/kg and comprising at least one polypeptide as defined above, or atleast one feed additive as defined above.

A composition comprising at least one polypeptide as defined above,together with at least one other enzyme selected from amongstalpha-amylase (EC 3.2.1.1), phytase (EC 3.1.3.8 or 3.1.3.26); xylanase(EC 3.2.1.8); galactanase (EC 3.2.1.89); alpha-galactosidase (EC3.2.1.22); protease (EC 3.4.−.−), phospholipase A1 (EC 3.1.1.32);phospholipase A2 (EC 3.1.1.4); lysophospholipase (EC 3.1.1.5);phospholipase C (3.1.4.3); phospholipase D (EC 3.1.4.4); and/orbeta-glucanase (EC 3.2.1.4 or EC 3.2.1.6).

Use of at least one polypeptide as defined above in detergents.

The embodiments of the above second, third, and fourth aspects, are,independently of each other, also preferred sub-aspects of the firstaspect of the invention, as well as preferred sub-aspects of each other.

DETAILED DESCRIPTION OF THE INVENTION

Polypeptides having protease activity, or proteases, are sometimes alsodesignated peptidases, proteinases, peptide hydrolases, or proteolyticenzymes. Proteases may be of the exo-type that hydrolyses peptidesstarting at either end thereof, or of the endo-type that act internallyin polypeptide chains (endopeptidases). Endopeptidases show activity onN- and C-terminally blocked peptide substrates that are relevant for thespecificity of the protease in question.

The term “protease” is defined herein as an enzyme that hydrolysespeptide bonds. It includes any enzyme belonging to the EC 3.4 enzymegroup (including each of the thirteen subclasses thereof. The EC numberrefers to Enzyme Nomenclature 1992 from NC-IUBMB, Academic Press, SanDiego, Calif., including supplements 1–5 published in Eur. J. Biochem.1994, 223,1–5; Eur. J. Biochem. 1995, 232, 1–6; Eur. J. Biochem. 1996,237, 1–5; Eur. J. Biochem. 1997, 250, 1–6; and Eur. J. Biochem. 1999,264, 610–650; respectively. The nomenclature is regularly supplementedand updated; see e.g. the World Wide Web (WWW) atwww.chem.qmw.ac.uk/iubmb/enzyme/index.html).

Proteases are classified on the basis of their catalytic mechanism intothe following groups: Serine proteases (S), Cysteine proteases (C),Aspartic proteases (A), Metalloproteases (M), and Unknown, or as yetunclassified, proteases (U), see Handbook of Proteolytic Enzymes, A. J.Barrett, N. D. Rawlings, J. F. Woessner (eds), Academic Press (1998), inparticular the general introduction part.

In particular embodiments, the proteases of the invention and for useaccording to the invention are selected from the group consisting of:

-   (a) Proteases belonging to the EC 3.4.−.− enzyme group;-   (b) Serine proteases belonging to the S group of the above Handbook;-   (c) Serine proteases of peptidase family S2A; and/or-   (d) Serine proteases of peptidase family S1E as described in    Biochem.J. 290:205–218 (1993) and in MEROPS protease database,    release 6.20, Mar. 24, 2003, (www.merops.ac.uk). The database is    described in Rawlings, N.D., O'Brien, E. A. & Barrett, A. J. (2002)    MEROPS: the protease database. Nucleic Acids Res. 30, 343–346.

For determining whether a given protease is a Serine protease, and afamily S2A protease, reference is made to the above Handbook and theprinciples indicated therein. Such determination can be carried out forall types of proteases, be it naturally occurring or wild-typeproteases; or genetically engineered or synthetic proteases.

Protease activity can be measured using any assay, in which a substrateis employed, that includes peptide bonds relevant for the specificity ofthe protease in question. Assay-pH and assay-temperature are likewise tobe adapted to the protease in question. Examples of assay-pH-Values arepH 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. Examples of assay-temperaturesare 30, 35, 37, 40, 45, 50, 55, 60, 65, 70, 80, 90, or 95° C.

Non-limiting examples of protease substrates are casein, such asAzurine-Crosslinked Casein (AZCL-casein), and haemoglobin. For thepurposes of determing specific activity of the protease of the inventionthe substrate is haemoglobin, and a suitable assay disclesed in Example3. Two other protease assays are described in Example 2, either of whichcan be used to determine protease activity in general. For purposesother than specific activity determinations, the so-called pNA Assay isa preferred assay.

The protease of the invention exhibits a specific activity onhaemoglobin at pH 7.5 and 25° C. of at least 39AU/g. The specificactivity may be determined as described in Example 3. The protease ofthe invention may exhibit a specific activity of at least 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, or at least 54 AU/g.

It is well-known that determination of specific activity includesdetermination of protein content, as well as protease activity, of thepurified protease.

In a particular embodiment, the protein content is determined by aminoacid analysis, for example by acid hydrolysis of the protease, andsubsequent separation and quantification of the released amino acids,preferably on a Biochrom 20 Plus Amino Acid Analyser.

The following are particular, optional, features of the proteaseactivity determination: (i) the haemoglobin substrate is denatured; (ii)the haemoglobin substrate is used in an amount of 0.65% w/w; (ii) theassay buffer is KH₂PO₄/NaOH buffer, pH 7.50; (ii) the reaction time forthe protease is 10 minutes; (iii) after the enzymatical reaction, theundigested haemoglobin is precipitated with trichloroacetic acid (TCA)and removed, preferably by filtration; (iv) the TCA-soluble haemoglobindegradation products in the filtrate are determined, preferably with theFolin & Ciocalteu's phenol reagent; (v) the activity unit (AU) ismeasured and defined by reference to an ALCALASE™ enzyme standard; (vi)the activity unit (AU) is measured using assay EB-SM-0349, preferablyEB-SM-0349 02/01; and/or (vii) the assay used is the “Protease ActivityAssay (AU/ml)” as disclosed in Example 3. The ALCALASE™ standard and theEB-SM-0349 assay is available from Novozymes A/S, Krogshoejvej 36,DK-2880 Bagsvaerd, Denmark (quoting “your ref. patent 10476” and“EB-SM-0349”, or “EB-SM-0349 02/01,” respectively).

A screening for proteases of high specific activity related to theproteases of SEQ ID NOs: 2 and 6 may be performed as follows: In a firststep, a DNA library is screened with primers, e.g. SEQ ID NO: 3, 4, 7,or 8, or preferably with the mature peptide encoding parts of either ofSEQ ID NO: 1 or 5; and hybridizing clones are expressed in a suitablestrain, e.g. a strain of Bacillus or E. coli. In a next step, theexpressed proteases related to SEQ ID NOs: 2 and/or 6 are purified,preferably in a micro-purification process (see e.g. WO 03/037914), andin a following step, the amount of active protease is determined foreach candidate by use of the well-known principle of active sitetitration (AST) with a strong inhibitor of the enzyme. This is with aview to being able to compare equal molar amounts of each protease inthe subsequent final step, which is a determination of the proteaseactivity of the now known amount of protease by any suitable assay, forexample the pNA assay of Example 2 herein. A major part of thisprocedure may be automatized, and if desired performed with theassistance of robots. The verification of high specific activity is e.g.done by purification of the protease, and establishment of the specificactivity as described in the experimental part herein.

There are no limitations on the origin of the protease of the inventionand/or for use according to the invention. Thus, the term proteaseincludes not only natural or wild-type proteases obtained frommicroorganisms of any genus, but also any mutants, variants, fragmentsetc. thereof exhibiting protease activity, as well as syntheticproteases, such as shuffled proteases, and consensus proteases. Suchgenetically engineered proteases can be prepared as is generally knownin the art, eg by Site-directed Mutagenesis, by PCR (using a PCRfragment containing the desired mutation as one of the primers in thePCR reactions), or by Random Mutagenesis. The preparation of consensusproteins is described in eg EP 897985. Gene shuffling is generallydescribed in e.g. WO 95/22625 and WO 96/00343. Recombination of proteasegenes can be made independently of the specific sequence of the parentsby synthetic shuffling as described in Ness, J. E. et al, in NatureBiotechnology, Vol. 20 (12), pp. 1251–1255, 2002. Syntheticoligonucleotides degenerated in their DNA sequence to provide thepossibility of all amino acids found in the set of parent proteases aredesigned and the genes assembled according to the reference. Theshuffling can be carried out for the full length sequence or for onlypart of the sequence and then later combined with the rest of the geneto give a full length sequence. Proteases having an amino acid sequencecomprising the mature parts of either of SEQ ID NO: 2 or 6 areparticular examples of such parent proteases which can be subjected toshuffling as described above, if desired together with, e.g., theprotease derived from Nocardiopsis sp. NRRL 18262, to provide additionalproteases of the invention. The term “obtained from” as used herein inconnection with a given source shall mean that the polypeptide encodedby the nucleic acid sequence is produced by the source or by a cell inwhich the nucleic acid sequence from the source is present. In apreferred embodiment, the polypeptide is secreted extracellularly.

In a specific embodiment, the protease is a low-allergenic variant,designed to invoke a reduced immunological response when exposed toanimals, including man. The term immunological response is to beunderstood as any reaction by the immune system of an animal exposed tothe protease. One type of immunological response is an allergic responseleading to increased levels of IgE in the exposed animal. Low-allergenicvariants may be prepared using techniques known in the art. For examplethe protease may be conjugated with polymer moieties shielding portionsor epitopes of the protease involved in an immunological response.Conjugation with polymers may involve in vitro chemical coupling ofpolymer to the protease, e.g. as described in WO 96/17929, WO 98/30682,WO 98/35026, and/or WO 99/00489. Conjugation may in addition oralternatively thereto involve in vivo coupling of polymers to theprotease. Such conjugation may be achieved by genetic engineering of thenucleotide sequence encoding the protease, inserting consensus sequencesencoding additional glycosylation sites in the protease and expressingthe protease in a host capable of glycosylating the protease, see e.g.WO 00/26354. Another way of providing low-allergenic variants is geneticengineering of the nucleotide sequence encoding the protease so as tocause the protease to self-oligomerize, effecting that protease monomersmay shield the epitopes of other protease monomers and thereby loweringthe antigenicity of the oligomers. Such products and their preparationis described e.g. in WO 96/16177. Epitopes involved in an immunologicalresponse may be identified by various methods such as the phage displaymethod described in WO 00/26230 and WO 01/83559, or the random approachdescribed in EP 561907. Once an epitope has been identified, its aminoacid sequence may be altered to produce altered immunological propertiesof the protease by known gene manipulation techniques such as sitedirected mutagenesis (see e.g. WO 00/26230, WO 00/26354 and/or WO00/22103) and/or conjugation of a polymer may be done in sufficientproximity to the epitope for the polymer to shield the epitope.

A polypeptide according to either aspect of the present invention maycomprise an amino acid sequence which has a degree of identity to themature peptide part of either of SEQ ID NO: 2 or 6, for example to aminoacids 1 to 188 of SEQ ID NO: 2, and/or to amino acids 1 to 192 of SEQ IDNO: 6 (the mature peptide parts), of, for example, at least about 65%,and which have protease activity (hereinafter “homologouspolypeptides”). In particular embodiments, the degree of identity toeither of the mature peptide parts of either of SEQ ID NO: 2 or 6, is atleast about 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%. In alternativeembodiments, the degree of identity is at least about 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, or at least about64%.

The present invention also relates to isolated polypeptides comprisingan amino acid sequence which has a degree of identity to amino acids−167 to 188 of SEQ ID NO: 2, and/or to amino acids −160 to 192 of SEQ IDNO: 6, of, for example, at least about 78%, and which have proteaseactivity. In particular embodiments, the degree of identity to aminoacids −167 to 188 of SEQ ID NO: 2, is at least about 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%.In alternative embodiments, the degree of identity is at least about50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, or at least 84%.

In particular embodiments, the polypeptides of the invention i) have; orii) consist of an amino acid sequence with any of the degrees ofidentity as mentioned above.

For the purposes of the present invention, the degree of identitybetween two amino acid sequences, as well as the degree of identitybetween two nucleotide sequences, is determined by the program “align”which is a Needleman-Wunsch alignment (i.e. a global alignment). Theprogram is used for alignment of polypeptide, as well as nucleotidesequences. The default scoring matrix BLOSUM50 is used for polypeptidealignments, and the default identity matrix is used for nucleotidealignments. The penalty for the first residue of a gap is −12 forpolypeptides and −16 for nucleotides. The penalties for further residuesof a gap are −2 for polypeptides, and −4 for nucleotide.

“Align” is part of the FASTA package version v20u6 (see W. R. Pearsonand D. J. Lipman (1988), “Improved Tools for Biological SequenceAnalysis”, PNAS 85:2444–2448, and W. R. Pearson (1990) “Rapid andSensitive Sequence Comparison with FASTP and FASTA,” Methods inEnzymology 183:63–98). FASTA protein alignments use the Smith-Watermanalgorithm with no limitation on gap size (see “Smith-Watermanalgorithm”, T. F. Smith and M. S. Waterman (1981) J. Mol. Biol.147:195–197).

In a particular embodiment, the mature peptide parts, or predicted orexpected mature peptide parts, of the two amino acid sequences are usedfor the alignment. In the alternative, that part of the sequence, whoseidentity to the mature peptide part of SEQ ID NO: 2 is being examined,is chosen, which according to a multiple alignment made as describedbelow is most similar to the mature peptide part of SEQ ID NO: 2, i.e.the corresponding amino acid residues as identified by the multiplealignment.

In the present context, the basis for numbering amino acid residues (orassigning position numbers, cf. the second aspect of the invention) isSEQ ID NO: 2 starting with A1 and ending with T188. In the alternative,the basis is amino acids 1–188 of the protease derived from Nocardiopsissp. NRRL 18262 (SEQ ID NO: 1 as disclosed in WO 01/58276, preferably SEQID NO: 1 as disclosed in WO 01/58276 in which A87 is substituted withT87). Proteases may comprise extensions as compared to the maturepeptide parts, viz. in the N-terminal, and/or the C-terminal endsthereof. The amino acids of such extensions, if any, are to be numberedas is usual in the art, i.e. for a C-terminal extension: 189, 190, 191and so forth, and for an N-terminal extension −1, −2, −3 and so forth.

For each amino acid residue in each protease aligned to the referencesequence, e.g. SEQ ID NO: 2, as explained above (for the purposes ofdetermining degree of identity), it is possible to directly andunambiguously assign an amino acid residue in the reference sequence,e.g. SEQ ID NO: 2, to which it corresponds. Corresponding residues areassigned the same position, or number, by reference to, e.g., SEQ ID NO:2.

For each amino acid residue in another protease, the correspondingposition of the reference sequence, e.g. SEQ ID NO: 2, can be found, asfollows:

The amino acid sequence of the other protease is designated SEQ X. Aposition corresponding to position N of SEQ ID NO: 2 is found asfollows: SEQ X is aligned with SEQ ID NO: 2 as specified above. From thealignment, the position in sequence SEQ X corresponding to position N ofSEQ ID NO: 2 can be clearly and unambiguously derived, using theprinciples described below.

SEQ X may be a mature part of the protease in question, or it may alsoinclude a signal peptide part, or it may be a fragment of the matureprotease which has protease activity, e.g. a fragment of the same lengthas SEQ ID NO: 2, and/or it may be the fragment which extends from A1toT188 when aligned with SEQ ID NO: 2 as described herein.

Three alignments are inserted below as Tables I, II and III. Thealignments were prepared as described above, aligning the mature part ofanother protease (SEQ X1, SEQ X2, and SEQ X3, respectively) to SEQ IDNO: 2. Approximately 50 amino acid residues of each protease are shown.

Looking first at the alignment of Table I, it is clear that, e.g., P42of SEQ ID NO: 2 corresponds to Q42 of SEQ X1, as these residues are ontop of each other in the alignment. They are both assigned number 42,viz. the number of the corresponding residue in SEQ ID NO: 2. It is alsoapparent from this alignment that, e.g., SEQ X1 does not comprise any of25S, 38T, 42P, 44S, or 49Q.

TABLE I ADIIGGLAYT MGGRCSVGFA ATNASGQPGF VTAGHCGTVG TPVSIGNGQG SEQ IDNO: 2 ADIIGGLAYT MGGRCSVGFA ATNAAGQPGF VTAGHCGRVG TQVTIGNGRG SEQ X1        10         20         30         40         50

Tables II and III are examples of alignments producing gaps in either ofthe two sequences.

In the alignment of Table II, a gap is produced in SEQ X2. Thehighlighted amino acid residue P of SEQ X2 is, for the present purposes,designated P28, although in SEQ X as such it is P25.

TABLE II ADIIGGLAYT MGGRCSVGFA ATNASGQPGF VTAGHCGTVG TPVSIGNGQG SEQ IDNO: 2 ADIIGGLAYT MGGRCSVGFA ATNA--- P GF VTAGHCGRVG TQVTIGNGRG SEQ X2        10         20         30         40         50

In the alignment of Table III, a gap is produced in SEQ X3. When a gapis produced between amino acids having position number nn and (nn+1) ofSEQ ID NO: 2, each position of the gap is assigned a lower case orsubscript letter: a, b, c etc. to the former position number, i.e. nn.Accordingly, each position of the gap is numbered nna, nnb etc. Thehighlighted amino acid residue R of SEQ X3 is, for the present purposes,designated R33a, although in SEQ X3 as such it is R34.

TABLE III ADIIGGLAYT MGGRCSVGFA ATNASGQPGF VTA--GHCGT VGTPVSIGNGQG SEQID NO: 2 ADIIGGLAYT MGGRCSVGFA ATNAAGQPGF VTA R SGHCGR VGTQVTIGNGRG SEQX3         10         20         30            40        50

In further particular embodiments of either aspect of the invention, thepolypeptide of the invention has a melting temperature Tm of at least75° C., 76° C., 77° C., 78° C., 79° C., 80° C., 81° C., 82° C., 83° C.,84° C., 85° C., 86° C., 87° C., 88° C., 89° C., 90° C., 91° C., 92° C.,93° C., 94° C., or at least 95° C., as determined by DifferentialScanning Calorimetry (DSC). The DSC is performed in a 10 mM sodiumphosphate, 50 mM sodium chloride buffer, pH 7.0. The scan rate isconstant, e.g. 1.5° C./min. The interval scanned may be from 20 to 100°C.

There are no upper limitations on the T_(m), however, it is presentlycontemplated that the T_(m) may be below 150° C., 145° C., 140° C., 135°C., 130° C., 125° C., 120° C., 115° C., 110° C., 105° C., or below 100°C.

In an alternative embodiment, another buffer is selected for thescanning, e.g. a buffer of pH 5.0, 5.5, 6.0, or pH 6.5.

In further alternative embodiments, a higher or lower scan rate may beused, e.g. a lower one of 1.4° C./min, 1.3° C./min, 1.2° C./min, 1.1°C./min, 1.0° C./min, or 0.9° C./min.

Reference is made to Example 2 for further details about the scanningprocedure.

In a particular embodiment, the protease of the invention exhibits anamended temperature activity profile as compared to the protease derivedfrom Nocardiopsis sp. NRRL 18262. For example, the protease of theinvention may exhibit a relative activity at pH 9 and 80° C. of at least0.40, preferably at least 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75,0.80, 0.85, 0.90, or at least 0.95, the term “relative” referring to themaximum activity measured for the protease in question. For the proteasederived from Nocardiopsis sp. NRRL 18262, the activity at 70° C. is setto 1.000 (100%), see Example 2. As another example, the protease of theinvention exhibits a relative activity at pH 9 and 90° C. of at least0.10, preferably at least 0.15, 0.20, 0.25, 0.30, or of at least 0.35.In a particular embodiment, the protease activity is measured using theProtazyme AK assay of Example 2.

The present invention also relates to the animal feed use of thepolypeptides of the invention.

The degree of identity between two amino acid sequences may also bedetermined by the Clustal method (Higgins, 1989, CABIOS 5: 151–153)using the LASERGENE™ MEGALIGN™ software (DNASTAR, Inc., Madison, Wis.)with an identity table and the following multiple alignment parameters:Gap penalty of 10, and gap length penalty of 10. Pairwise alignmentparameters are Ktuple=1, gap penalty=3, windows=5, and diagonals=5. Thedegree of identity between two nucleotide sequences may be determinedusing the same algorithm and software package as described above withthe following settings: Gap penalty of 10, and gap length penalty of 10.Pairwise alignment parameters are Ktuple=3, gap penalty=3 andwindows=20.

In a particular embodiment, the homologous polypeptides have an aminoacid sequence that differs from (a) the mature peptide parts of eitherof SEQ ID NO: 2 or 6, or (b) from the proforms thereof (excluding signalpeptide parts, including mature peptide parts), by (i) no more than 50,49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32,31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, or no more than 20 aminoacids; (ii) no more than twenty, nineteen, eighteen, seventeen, sixteen,fifteen, fourteen, thirteen, twelve, or no more than eleven amino acids;(iii) no more than ten, nine, eight, seven, six, five, four, three, two,or no more than one amino acid; (iv) ten, or by nine, or by eight, or byseven, or by six, or by five amino acids; or (v) four, or by three, orby two amino acids, or by one amino acid.

In a particular embodiment, the polypeptides of the present inventioncomprise the amino acid sequence of the mature peptide part of either ofSEQ ID NO: 2 or 6, or allelic variants thereof; or fragments thereofthat have protease activity.

In another preferred embodiment, the polypeptides of the presentinvention consist of the mature peptide part of either of SEQ ID NO: 2or 6, or allelic variants thereof; or fragments thereof that haveprotease activity.

A fragment is a polypeptide having one or more amino acids deleted fromthe amino and/or carboxyl terminus of these amino acid sequences. In oneembodiment a fragment contains at least 75 amino acid residues, or atleast 100 amino acid residues, or at least 125 amino acid residues, orat least 150 amino acid residues, or at least 160 amino acid residues,or at least 165 amino acid residues, or at least 170 amino acidresidues, or at least 175 amino acid residues.

An allelic variant denotes any of two or more alternative forms of agene occupying the same chromosomal locus. Allelic variation arisesnaturally through mutation, and may result in polymorphism withinpopulations. Gene mutations can be silent (no change in the encodedpolypeptide) or may encode polypeptides having altered amino acidsequences. An allelic variant of a polypeptide is a polypeptide encodedby an allelic variant of a gene.

The present invention also relates to isolated polypeptides havingprotease activity and which are encoded by nucleic acid sequences whichhybridize under very low, or low, or medium, or medium-high, or high, orvery high stringency conditions with a nucleic acid probe whichhybridizes under the same conditions with (a) nucleotides 1–1065,preferably 502–1065, or of SEQ ID NO: 1, and/or nucleotides 1–1143,preferably 568–1143 of SEQ ID NO: 5; (b) a subsequence of (a), or (c) acomplementary strand of (a), or (b) (J. Sambrook, E. F. Fritsch, and T.Maniatis, 1989, Molecular Cloning, A Laboratory Manual, 2nd edition,Cold Spring Harbor, N.Y.). In one particular embodiment the nucleic acidprobe is selected from amongst the nucleic acid sequences of (a), (b),or (c) above.

The subsequence of the nucleotides mentioned under (a) above may be atleast 100 nucleotides, or in another embodiment at least 200nucleotides. Moreover, the subsequence may encode a polypeptide fragmentthat has protease activity.

The nucleic acid sequences of (a) above, or a subsequence thereof, aswell as the corresponding parts of the amino acid sequence of SEQ ID NO:2 or 6, or a fragment thereof, may be used to design a nucleic acidprobe to identify and clone DNA encoding polypeptides having proteaseactivity from strains of different genera or species according tomethods well known in the art. In particular, such probes can be usedfor hybridization with the genomic or cDNA of the genus or species ofinterest, following standard Southern blotting procedures, in order toidentify and isolate the corresponding gene therein. Such probes can beconsiderably shorter than the entire sequence, but should be at least15, preferably at least 25, and more preferably at least 35 nucleotidesin length. Longer probes can also be used. Both DNA and RNA probes canbe used. The probes are typically labeled for detecting thecorresponding gene (for example, with ³²P, ³H, ³⁵S, biotin, or avidin).Such probes are encompassed by the present invention.

Thus, a genomic DNA or cDNA library prepared from such other organismsmay be screened for DNA that hybridizes with the probes described aboveand which encodes a polypeptide having protease activity. Genomic orother DNA from such other organisms may be separated by agarose orpolyacrylamide gel electrophoresis, or other separation techniques. DNAfrom the libraries or the separated DNA may be transferred to andimmobilized on nitrocellulose or other suitable carrier material. Inorder to identify a clone or DNA which is homologous with either of SEQID NO: 1 or 5, or a subsequence thereof, the carrier material is used ina Southern blot. For purposes of the present invention, hybridizationindicates that the nucleic acid sequence hybridizes to a labeled nucleicacid probe corresponding to the nucleic acid sequence shown in either ofSEQ ID NO: 1 or 5; the complementary strands thereof, or subsequencesthereof, under very low to very high stringency conditions. Molecules towhich the nucleic acid probe hybridizes under these conditions aredetected using X-ray film.

For long probes of at least 100 nucleotides in length, very low to veryhigh stringency conditions are defined as prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and either 25% formamide for very lowand low stringencies, 35% formamide for medium and medium-highstringencies, or 50% formamide for high and very high stringencies,following standard Southern blotting procedures.

For long probes of at least 100 nucleotides in length, the carriermaterial is finally washed three times each for 15 minutes using0.2×SSC, 0.2% SDS, 20% formamide preferably at least at 45° C. (very lowstringency), more preferably at least at 50° C. (low stringency), morepreferably at least at 55° C. (medium stringency), more preferably atleast at 60° C. (medium-high stringency), even more preferably at leastat 65° C.(high stringency), and most preferably at least at 70° C. (veryhigh stringency).

For short probes about 15 nucleotides to about 70 nucleotides in length,stringency conditions are defined as prehybridization, hybridization,and washing post-hybridization at 5° C. to 10° C. below the calculatedT_(m) using the calculation according to Bolton and McCarthy (1962,Proceedings of the National Academy of Sciences USA 48:1390) in 0.9 MNaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA, 0.5% NP-40, 1× Denhardt'ssolution, 1 mM sodium pyrophosphate, 1 mM sodium monobasic phosphate,0.1 mM ATP, and 0.2 mg of yeast RNA per ml following standard Southernblotting procedures.

For short probes about 15 nucleotides to about 70 nucleotides in length,the carrier material is washed once in 6×SSC plus 0.1% SDS for 15minutes and twice each for 15 minutes using 6×SSC at 5° C. to 10° C.below the calculated T_(m).

The present invention also relates to variants of the polypeptide havingan amino acid sequence of the mature part of either of SEQ ID NO: 2 or6, comprising a substitution, deletion, and/or insertion of one or moreamino acids.

The amino acid sequences of the variant polypeptides may differ from theamino acid sequence of the mature part of either of SEQ ID NO: 2 or 6,by an insertion or deletion of one or more amino acid residues and/orthe substitution of one or more amino acid residues by different aminoacid residues. In a particular embodiment, amino acid changes are of aminor nature, that is conservative amino acid substitutions that do notsignificantly affect the folding and/or activity of the protein; smalldeletions, typically of one to about 30 amino acids; small amino- orcarboxyl-terminal extensions, such as an amino-terminal methionineresidue; a small peptide of up to about 20–25 residues; or a smallextension that facilitates purification by changing net charge oranother function, such as a poly-histidine tract, an antigenic epitopeor a binding domain.

Examples of conservative substitutions are within the group of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine).Accordingly, for example, the invention relates to a polypeptide having,or comprising, a sequence as set forth in either of SEQ ID NO: 2 or 6,preferably the mature parts thereof, wherein conservative amino acidsubstitutions comprise replacements, one for another, among the basicamino acids (arginine, lysine and histidine), among the acidic aminoacids (glutamic acid and aspartic acid), among the polar amino acids(glutamine and asparagine), among the hydrophobic amino acids (alanine,leucine, isoleucine, and valine), among the aromatic amino acids(phenylalanine, tryptophan and tyrosine), and among the small aminoacids (glycine, alanine, serine, threonine and methionine), or anycombination thereof, or active fragments thereof.

Amino acid substitutions which do not generally alter the specificactivity are known in the art and are described, for example, by H.Neurath and R. L. Hill, 1979, In, The Proteins, Academic Press, NewYork. The most commonly occurring exchanges are Ala/Ser, Val/Ile,Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe,Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly aswell as these in reverse.

In a particular embodiment, the polypeptides of the invention and foruse according to the invention are acid-stable. For the presentpurposes, the term acid-stable means that the residual activity after 2hours of incubation at pH 2.0, pH 2.5 or pH 3.0 and 37° C., is at least50%, as compared to the residual activity of a corresponding sampleincubated for 2 hours at pH 9.0 and 5° C. In a particular embodiment,the residual activity is at least 60%, 70%, 80% or at least 90%. Asuitable assay for determining acid-stability is the pH-stability assayof Example 2.

In another particular embodiment, the polypeptides of the invention andfor use according to the invention have a relative activity at pH 7.0 ofat least 0.10, 0.15, 0.20, 0.25, 0.30 or at least 0.35. The pH-profiletest of Example 2 is used for the determination.

In still further particular embodiments, the polypeptides of theinvention and for use or at least 0.20; and/or ii) a relative activityat 70° C. of at least 0.40, 0.50, or at least 0.56. Thetemperature-profile test of Example 2 is used for these determinations.

In still further particular embodiments, the polypeptides of theinvention and for use according to the invention have a T_(m), asdetermined by DSC, of at least 78° C. or of at least 79, 80, 81, 82, orof at least 83° C. T_(m) is determined at pH 7.0 as described in Example2.

The polypeptide of the invention and for use according to the inventionmay be a bacterial or fungal polypeptide. The fungal polypeptide may bederived from a filamentous fungus or from a yeast.

In particular embodiments, the polypeptide of the invention is i) abacterial protease; ii) a protease of the phylum Actinobacteria; iii) ofthe class Actinobacteria; iv) of the order Actinomycetales v) of thefamily Nocardiopsaceae; vi) of the genus Nocardiopsis; and/or a proteasederived from vii) Nocardiopsis species such as, Nocardiopsisdassonvillei, Nocardiopsis alkaliphila, Nocardiopsis antarctica,Nocardiopsis prasina, Nocardiopsis composta, Nocardiopsis exhalans,Nocardiopsis halophila, Nocardiopsis halotolerans, Nocardiopsiskunsanensis, Nocardiopsis listeri, Nocardiopsis lucentensis,Nocardiopsis metallicus, Nocardiopsis synnemataformans, Nocardiopsistrehalosi, Nocardiopsis tropica, Nocardiopsis umidischolae, Nocardiopsisxinjiangensis, or Nocardiopsis alba, for example Nocardiopsis alba DSM15647, such as a polypeptide with the amino acid sequence of amino acids1 to 188 or −167 to 188 of SEQ ID NO: 2, or from Nocardiopsisdassonvillei subsp. dassonvillei DSM 4235, such as a polypeptide withthe amino acid sequence of amino acids 1–192, or −160 to 192 of SEQ IDNO: 6. In a particular embodiment, the protease derives fromNocardiopsis alba.

The above taxonomy is according to the chapter: The road map to theManual by G. M. Garrity & J. G. Holt in Bergey's Manual of SystematicBacteriology, 2001, second edition, volume 1, David R. Bone, Richard W.Castenholz.

It will be understood that for the aforementioned species, the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), andAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL). E.g., Nocardiopsis dassonvillei subsp.dassonvillei DSM 43235 is publicly available from DSMZ (DeutscheSammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig,Germany). This strain was also deposited at other depositaryinstitutions as follows: ATCC 23219, IMRU 1250, NCTC 10489.

Furthermore, such polypeptides may be identified and obtained from othersources including microorganisms isolated from nature (e.g., soil,composts, water, etc.) using the above-mentioned probes. Techniques forisolating microorganisms from natural habitats are well known in theart. The nucleic acid sequence may then be derived by similarlyscreening a genomic or cDNA library of another microorganism. Once anucleic acid sequence encoding a polypeptide has been detected with theprobe(s), the sequence may be isolated or cloned by utilizing techniqueswhich are known to those of ordinary skill in the art (see, e.g.,Sambrook et al., 1989, supra).

As defined herein, an “isolated,” or “purified,” polypeptide is apolypeptide which is essentially free of other polypeptides, e.g., atleast about 20% pure, preferably at least about 40% pure, morepreferably about 60% pure, even more preferably about 80% pure, mostpreferably about 90% pure, and even most preferably about 95% pure, asdetermined by SDS-PAGE.

Polypeptides encoded by nucleic acid sequences of the present inventionalso include fused polypeptides or cleavable fusion polypeptides inwhich another polypeptide is fused at the N-terminus or the C-terminusof the polypeptide or fragment thereof. A fused polypeptide is producedby fusing a nucleic acid sequence (or a portion thereof) encodinganother polypeptide to a nucleic acid sequence (or a portion thereof) ofthe present invention. Techniques for producing fusion polypeptides areknown in the art, e.g. PCR, or ligating the coding sequences encodingthe polypeptides so that they are in frame and that expression of thefused polypeptide is under control of the same promoter(s) andterminator.

In still further particular embodiments, the invention excludes one ormore of the proteases derived from (i) Nocardiopsis dassonvillei NRRL18133 which is disclosed in WO 88/03947; (ii) Nocardiopsis sp. strainOPC-210 (FERM P-10508) which is disclosed in JP 2-255081-A; (iii) strainZIMET 43647 of the species Nocardiopsis dassonvillei which is disclosedin DD 200432|8; (iv) Nocardiopsis sp. TOA-1 (FERM-P-18676) which isdisclosed in JP 2003284571; and/or (v) the corresponding DNA.

Nucleic Acid Sequences The present invention also relates to isolatednucleic acid sequences that encode a polypeptide of the presentinvention. Particular nucleic acid sequences of the invention arenucleotides 502–1065, or 1–1065, of SEQ ID NO: 1, of which nucleotides502–1065 of SEQ ID NO: 1, correspond to the mature polypeptide encodingregion, as well as nucleotides 1–1143, preferably 568–1143, of SEQ IDNO: 5. The present invention also encompasses nucleic acid sequenceswhich encode a polypeptide having the amino acid sequence of amino acids−167 to 188, preferably 1–188, of SEQ ID NO: 2, or amino acids −160 to192, preferably 1–192, of SEQ ID NO: 6, which differ from thecorresponding parts of SEQ ID NO: 1 by virtue of the degeneracy of thegenetic code. The present invention also relates to subsequences of SEQID NOs: 1 and 5, which encode fragments of SEQ ID NOs: 2 and 6,respectively, which have protease activity.

A subsequence of either of SEQ ID NO: 1 or 5 is a nucleic acid sequenceencompassed by either of SEQ ID NO: 1 or 5, except that one or morenucleotides from the 5′ and/or 3′ end has been deleted. Preferably, asubsequence contains at least 225 nucleotides, more preferably at least300 nucleotides, even more preferably at least 375, 450, 500, 531, 600,700, 800, 900 or 1000 nucleotides.

The present invention also relates to nucleotide sequences which have adegree of identity to (i) nucleotides 502–1065 of SEQ ID NO: 1, and/ornucleotides 568–1143 of SEQ ID NO: 5, or to (ii) nucleotides 1–1065 ofSEQ ID NO: 1, and/or nucleotides 88–1143 of SEQ ID NO: 5, of at least74%. In particular embodiments, the degree of identity to either of thenucleotides of (i) or (ii) is at least 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or at least 99%. In other particularembodiments, the degree of identity to either of the nucleotides of (i)or (ii) is at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,70%, 71%, 72%, or at least 73%.

The present invention also relates to mutant nucleic acid sequencescomprising at least one mutation in either of SEQ ID NO: 1 or 5,preferably in the mature peptide encoding parts, in which the mutantnucleic acid sequence encodes a polypeptide which (i) consists of aminoacids −167 to 188, preferably 1–188, of SEQ ID NO: 2, or amino acids−160 to 192, preferably 1–192, of SEQ ID NO: 6; or (ii) is a variant ofany of the sequences of (i), wherein the variant comprises asubstitution, deletion, and/or insertion of one or more amino acids, or(iii) is an allelic variant of any of the sequences of (i), or (iv) is afragment of any of the sequences of (i).

The techniques used to isolate or clone a nucleic acid sequence encodinga polypeptide are known in the art and include isolation from genomicDNA, preparation from cDNA, or a combination thereof. The cloning of thenucleic acid sequences of the present invention from such genomic DNAcan be effected, e.g., by using the well known polymerase chain reaction(PCR) or antibody screening of expression libraries to detect cloned DNAfragments with shared structural features. See, e.g., Innis et al.,1990, PCR: A Guide to Methods and Application, Academic Press, New York.Other nucleic acid amplification procedures such as ligase chainreaction (LCR), ligated activated transcription (LAT) and nucleic acidsequence-based amplification (NASBA) may be used. The nucleic acidsequence may be cloned from a strain of Nocardiopsis or another orrelated organism and thus, for example, may be an allelic or speciesvariant of the polypeptide encoding region of the nucleic acid sequence.

The term “isolated nucleic acid sequence” as used herein refers to anucleic acid sequence which is essentially free of other nucleic acidsequences, e.g., at least about 20% pure, preferably at least about 40%pure, more preferably at least about 60% pure, even more preferably atleast about 80% pure, and most preferably at least about 90% pure asdetermined by agarose electrophoresis. For example, an isolated nucleicacid sequence can be obtained by standard cloning procedures used ingenetic engineering to relocate the nucleic acid sequence from itsnatural location to a different site where it will be reproduced. Thecloning procedures may involve excision and isolation of a desirednucleic acid fragment comprising the nucleic acid sequence encoding thepolypeptide, insertion of the fragment into a vector molecule, andincorporation of the recombinant vector into a host cell where multiplecopies or clones of the nucleic acid sequence will be replicated. Thenucleic acid sequence may be of genomic, cDNA, RNA, semisynthetic,synthetic origin, or any combinations thereof.

Modification of a nucleic acid sequence encoding a polypeptide of thepresent invention may be necessary for the synthesis of polypeptidessubstantially similar to the polypeptide. The term “substantiallysimilar” to the polypeptide refers to non-naturally occurring forms ofthe polypeptide. These polypeptides may differ in some engineered wayfrom the polypeptide isolated from its native source, e.g., variantsthat differ in specific activity, thermostability, pH optimum,allergenicity, or the like. The variant sequence may be constructed onthe basis of the nucleic acid sequence presented as the polypeptideencoding part of SEQ ID NOs: 1 and/or 5, e.g., a subsequence thereof,and/or by introduction of nucleotide substitutions which do not giverise to another amino acid sequence of the polypeptide encoded by thenucleic acid sequence, but which correspond to the codon usage of thehost organism intended for production of the protease, or byintroduction of nucleotide substitutions which may give rise to adifferent amino acid sequence. For a general description of nucleotidesubstitution, see, e.g., Ford et al., 1991, Protein Expression andPurification 2: 95–107. Low-allergenic polypeptides can e.g. be preparedas described above.

It will be apparent to those skilled in the art that such substitutionscan be made outside the regions critical to the function of the moleculeand still result in an active polypeptide. Amino acid residues essentialto- the activity of the polypeptide encoded by the isolated nucleic acidsequence of the invention, and therefore preferably not subject tosubstitution, may be identified according to procedures known in theart, such as site-directed mutagenesis or alanine-scanning mutagenesis(see, e.g., Cunningham and Wells, 1989, Science 244: 1081–1085). In thelatter technique, mutations are introduced at every positively chargedresidue in the molecule, and the resultant mutant molecules are testedfor protease activity to identify amino acid residues that are criticalto the activity of the molecule. Sites of substrate-protease interactioncan also be determined by analysis of the three-dimensional structure asdetermined by such techniques as nuclear magnetic resonance analysis,crystallography or photoaffinity labelling (see, e.g., de Vos et al.,1992, Science 255: 306–312; Smith et al., 1992, Journal of MolecularBiology 224: 899–904; Wlodaver et al., 1992, FEBS Letters 309: 59–64).

The present invention also relates to isolated nucleic acid sequencesencoding a polypeptide of the present invention, which hybridize undervery low stringency conditions, preferably low stringency conditions,more preferably medium stringency conditions, more preferablymedium-high stringency conditions, even more preferably high stringencyconditions, and most preferably very high stringency conditions with anucleic acid probe which hybridizes under the same conditions with thenucleic acid sequence of SEQ ID NO: 1 and/or 5, or their complementarystrands; or allelic variants and subsequences thereof (Sambrook et al.,1989, supra), as defined herein.

The present invention also relates to isolated nucleic acid sequencesproduced by (a) hybridizing a DNA under very low, low, medium,medium-high, high, or very high stringency conditions with (i)nucleotides 1–1065, preferably 502–1065, of SEQ ID NO: 1, or nucleotides1–1143, or 88–1143, preferably 568–1143 of SEQ ID NO: 5, (ii) asubsequence of (i), or (iii) a complementary strand of (i), or (ii); and(b) isolating the nucleic acid sequence. The subsequence is preferably asequence of at least 100 nucleotides such as a sequence that encodes apolypeptide fragment which has protease activity.

Methods for Producing Mutant Nucleic Acid Sequences

The present invention further relates to methods for producing a mutantnucleic acid sequence, comprising introducing at least one mutation intothe mature polypeptide coding sequence of SEQ ID NOs: 1 and/or 5, or asubsequence thereof, wherein the mutant nucleic acid sequence encodes apolypeptide which consists of amino acids −167 to 188, preferably 1–188,of SEQ ID NO: 2, or amino acids −160 to 192, preferably 1–192, of SEQ IDNO: 6; or a fragment thereof which has protease activity.

The introduction of a mutation into the nucleic acid sequence toexchange one nucleotide for another nucleotide may be accomplished bysite-directed mutagenesis using any of the methods known in the art.Particularly useful is the procedure that utilizes a supercoiled, doublestranded DNA vector with an insert of interest and two synthetic primerscontaining the desired mutation. The oligonucleotide primers, eachcomplementary to opposite strands of the vector, extend duringtemperature cycling by means of Pfu DNA polymerase. On incorporation ofthe primers, a mutated plasmid containing staggered nicks is generated.Following temperature cycling, the product is treated with Dpnl which isspecific for methylated and hemimethylated DNA to digest the parentalDNA template and to select for mutation-containing synthesized DNA.Other procedures known in the art may also be used.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga nucleic acid sequence of the present invention operably linked to oneor more control sequences that direct the expression of the codingsequence in a suitable host cell under conditions compatible with thecontrol sequences. Expression will be understood to include any stepinvolved in the production of the polypeptide including, but not limitedto, transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

“Nucleic acid construct” is defined herein as a nucleic acid molecule,either single- or double-stranded, which is isolated from a naturallyoccurring gene or which has been modified to contain segments of nucleicacid combined and juxtaposed in a manner that would not otherwise existin nature. The term nucleic acid construct is synonymous with the termexpression cassette when the nucleic acid construct contains all thecontrol sequences required for expression of a coding sequence of thepresent invention. The term “coding sequence” is defined herein as anucleic acid sequence that directly specifies the amino acid sequence ofits protein product. The boundaries of the coding sequence are generallydetermined by a ribosome binding site (prokaryotes) or by the ATG startcodon (eukaryotes) located just upstream of the open reading frame atthe 5′ end of the mRNA and a transcription terminator sequence locatedjust downstream of the open reading frame at the 3′ end of the mRNA. Acoding sequence can include, but is not limited to, DNA, cDNA, andrecombinant nucleic acid sequences.

An isolated nucleic acid sequence encoding a polypeptide of the presentinvention may be manipulated in a variety of ways to provide forexpression of the polypeptide. Manipulation of the nucleic acid sequenceprior to its insertion into a vector may be desirable or necessarydepending on the expression vector. The techniques for modifying nucleicacid sequences utilizing recombinant DNA methods are well known in theart.

The term “control sequences” is defined herein to include all componentsthat are necessary or advantageous for the expression of a polypeptideof the present invention. Each control sequence may be native or foreignto the nucleic acid sequence encoding the polypeptide. Such controlsequences include, but are not limited to, a leader, polyadenylationsequence, propeptide sequence, promoter, signal peptide sequence, andtranscription terminator. At a minimum, the control sequences include apromoter, and transcriptional and translational stop signals. Thecontrol sequences may be provided with linkers for the purpose ofintroducing specific restriction sites facilitating ligation of thecontrol sequences with the coding region of the nucleic acid sequenceencoding a polypeptide. The term “operably linked” is defined herein asa configuration in which a control sequence is appropriately placed at aposition relative to the coding sequence of the DNA sequence such thatthe control sequence directs the expression of a polypeptide.

The control sequence may be an appropriate promoter sequence, a nucleicacid sequence that is recognized by a host cell for expression of thenucleic acid sequence. The promoter sequence contains transcriptionalcontrol sequences that mediate the expression of the polypeptide. Thepromoter may be any nucleic acid sequence which shows transcriptionalactivity in the host cell of choice including mutant, truncated, andhybrid promoters, and may be obtained from genes encoding extracellularor intracellular polypeptides either homologous or heterologous to thehost cell.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention, especially in abacterial host cell, are the promoters obtained from the E. coli lacoperon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilislevansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM),Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacilluslicheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylBgenes, and prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978,Proceedings of the National Academy of Sciences USA 75: 3727–3731), aswell as the tac promoter (DeBoer et al., 1983, Proceedings of theNational Academy of Sciences USA 80: 21–25). Further promoters aredescribed in “Useful proteins from recombinant bacteria” in ScientificAmerican, 1980, 242: 74–94; and in Sambrook et al., 1989, supra.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus oryzaeTAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus nigerneutral alpha-amylase, Aspergillus niger acid stable alpha-amylase,Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucormiehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzaetriose phosphate isomerase, Aspergillus nidulans acetamidase, andFusarium oxysporum trypsin-like protease (WO 96/00787), as well as theNA2-tpi promoter (a hybrid of the promoters from the genes forAspergillus niger neutral alpha-amylase and Aspergillus oryzae triosephosphate isomerase), and mutant, truncated, and hybrid promotersthereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP), andSaccharomyces cerevisiae 3-phosphoglycerate kinase. Other usefulpromoters for yeast host cells are described by Romanos et al., 1992,Yeast 8: 423–488.

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleic acid sequence encoding the polypeptide. Anyterminator which is functional in the host cell of choice may be used inthe present invention.

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus oryzae TAKA amylase, Aspergillus nigerglucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillusniger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

Preferred terminators for bacterial host cells, such as a Bacillus hostcell, are the terminators from Bacillus licheniformis alpha-amylase gene(amyL), the Bacillus stearothermophilus maltogenic amylase gene (amyM),or the Bacillus amyloliquefaciens alpha-amylase gene (amyQ).

The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA which is important for translation bythe host cell. The leader sequence is operably linked to the 5′ terminusof the nucleic acid sequence encoding the polypeptide. Any leadersequence that is functional in the host cell of choice may be used inthe present invention.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′ terminus of the nucleic acid sequence andwhich, when transcribed, is recognized by the host cell as a signal toadd polyadenosine residues to transcribed mRNA. Any polyadenylationsequence which is functional in the host cell of choice may be used inthe present invention.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus oryzae TAKA amylase,Aspergillus niger glucoamylase, Aspergillus nidulans anthranilatesynthase, Fusarium oxysporum trypsin-like protease, and Aspergillusniger alpha-glucosidase.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Molecular Cellular Biology 15: 5983–5990.

The control sequence may also be a signal peptide coding region thatcodes for an amino acid sequence linked to the amino terminus of apolypeptide and directs the encoded polypeptide into the cell'ssecretory pathway. The 5′ end of the coding sequence of the nucleic acidsequence may inherently contain a signal peptide coding region naturallylinked in translation reading frame with the segment of the codingregion which encodes the secreted polypeptide. Alternatively, the 5′ endof the coding sequence may contain a signal peptide coding region whichis foreign to the coding sequence. The foreign signal peptide codingregion may be required where the coding sequence does not naturallycontain a signal peptide coding region. Alternatively, the foreignsignal peptide coding region may simply replace the natural signalpeptide coding region in order to enhance secretion of the polypeptide.However, any signal peptide coding region which directs the expressedpolypeptide into the secretory pathway of a host cell of choice may beused in the present invention.

Effective signal peptide coding regions for bacterial host cells are thesignal peptide coding regions obtained from the genes for Bacillus NCIB11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase,Bacillus licheniformis subtilisin, Bacillus licheniformis alpha-amylase,Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), andBacillus subtilis prsA. Further signal peptides are described by Simonenand Palva, 1993, Microbiological Reviews 57: 109–137.

Effective signal peptide coding regions for filamentous fungal hostcells are the signal peptide coding regions obtained from the genes forAspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase,Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase,Humicola insolens cellulase, and Humicola lanuginosa lipase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding regions are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding region that codesfor an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide is known as a proenzyme orpropolypeptide (or a zymogen in some cases). A propolypeptide isgenerally inactive and can be converted to a mature active polypeptideby catalytic or autocatalytic cleavage of the propeptide from thepropolypeptide. The propeptide coding region may be obtained from thegenes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilisneutral protease (nprT), Saccharomyces cerevisiae alpha-factor,Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophilalaccase (WO 95/33836).

In a preferred embodiment, the propeptide coding region is nucleotides1–501 of SEQ ID NO: 1 or nucleotides 88–567 of SEQ ID NO: 5.

Where both signal peptide and propeptide regions are present at theamino terminus of a polypeptide, the propeptide region is positionednext to the amino terminus of a polypeptide and the signal peptideregion is positioned next to the amino terminus of the propeptideregion.

It may also be desirable to add regulatory sequences which allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those which causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. Regulatory systems in prokaryotic systems include the lac,tac, and trp operator systems. In yeast, the ADH2 system or GAL1 systemmay be used. In filamentous fungi, the TAKA alpha-amylase promoter,Aspergillus niger glucoamylase promoter, and Aspergillus oryzaeglucoamylase promoter may be used as regulatory sequences. Otherexamples of regulatory sequences are those which allow for geneamplification. In eukaryotic systems, these include the dihydrofolatereductase gene which is amplified in the presence of methotrexate, andthe metallothionein genes which are amplified with heavy metals. Inthese cases, the nucleic acid sequence encoding the polypeptide would beoperably linked with the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a nucleic acid sequence of the present invention, a promoter,and transcriptional and translational stop signals. The various nucleicacid and control sequences described above may be joined together toproduce a recombinant expression vector which may include one or moreconvenient restriction sites to allow for insertion or substitution ofthe nucleic acid sequence encoding the polypeptide at such sites.Alternatively, the nucleic acid sequence of the present invention may beexpressed by inserting the nucleic acid sequence or a nucleic acidconstruct comprising the sequence into an appropriate vector forexpression. In creating the expression vector, the coding sequence islocated in the vector so that the coding sequence is operably linkedwith the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) which can be conveniently subjected to recombinant DNA proceduresand can bring about the expression of the nucleic acid sequence. Thechoice of the vector will typically depend on the compatibility of thevector with the host cell into which the vector is to be introduced. Thevectors may be linear or closed circular plasmids.

The vector may be an autonomously replicating vector, i.e., a vectorwhich exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one which, when introduced into thehost cell, is integrated into the genome and replicated together withthe chromosome(s) into which it has been integrated. Furthermore, asingle vector or plasmid or two or more vectors or plasmids whichtogether contain the total DNA to be introduced into the genome of thehost cell, or a transposon may be used.

The vectors of the present invention preferably contain one or moreselectable markers which permit easy selection of transformed cells. Aselectable marker is a gene the product of which provides for biocide orviral resistance, resistance to heavy metals, prototrophy to auxotrophs,and the like. Examples of bacterial selectable markers are the dal genesfrom Bacillus subtilis or Bacillus licheniformis. Suitable markers foryeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3.Selectable markers for use in a filamentous fungal host cell include,but are not limited to, amdS (acetamidase), argB (ornithinecarbamoyltransferase), bar (phosphinothricin acetyltransferase), hygB(hygromycin phosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),trpC (anthranilate synthase), as well as equivalents thereof. Preferredfor use in an Aspergillus cell are the amdS and pyrG genes ofAspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus.

The vectors of the present invention preferably contain an element(s)that permits stable integration of the vector into the host cell'sgenome or autonomous replication of the vector in the cell independentof the genome.

For integration into the host cell genome, the vector may rely on thenucleic acid sequence encoding the polypeptide or any other element ofthe vector for stable integration of the vector into the genome byhomologous or nonhomologous recombination. Alternatively, the vector maycontain additional nucleic acid sequences for directing integration byhomologous recombination into the genome of the host cell. Theadditional nucleic acid sequences enable the vector to be integratedinto the host cell genome at a precise location(s) in the chromosome(s).To increase the likelihood of integration at a precise location, theintegrational elements should preferably contain a sufficient number ofnucleic acids, such as 100 to 1,500 base pairs, preferably 400 to 1,500base pairs, and most preferably 800 to 1,500 base pairs, which arehighly homologous with the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding nucleic acid sequences. On the other hand, thevector may be integrated into the genome of the host cell bynon-homologous recombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. Examples of bacterial origins of replication are theorigins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184permitting replication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1permitting replication in Bacillus. Examples of origins of replicationfor use in a yeast host cell are the 2 micron origin of replication,ARS1, ARS4, the combination of ARS1 and CEN3, and the combination ofARS4 and CEN6. The origin of replication may be one having a mutationwhich makes it functioning temperature-sensitive in the host cell (see,e.g., Ehrlich, 1978, Proceedings of the National Academy of Sciences USA75: 1433).

More than one copy of a nucleic acid sequence of the present inventionmay be inserted into the host cell to increase production of the geneproduct. An increase in the copy number of the nucleic acid sequence canbe obtained by integrating at least one additional copy of the sequenceinto the host cell genome or by including an amplifiable selectablemarker gene with the nucleic acid sequence where cells containingamplified copies of the selectable marker gene, and thereby additionalcopies of the nucleic acid sequence, can be selected for by cultivatingthe cells in the presence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

The protease may also be co-expressed together with at least one otherenzyme of interest for animal feed, such as an amylase, for examplealpha-amylase (EC 3.2.1.1), phytase (EC 3.1.3.8 or 3.1.3.26); xylanase(EC 3.2.1.8); galactanase (EC 3.2.1.89); alpha-galactosidase (EC3.2.1.22); protease (EC 3.4.−.−), phospholipase Al (EC 3.1.1.32);phospholipase A2 (EC 3.1.1.4); lysophospholipase (EC 3.1.1.5);phospholipase C (3.1.4.3); phospholipase D (EC 3.1.4.4); and/orbeta-glucanase (EC 3.2.1.4 or EC 3.2.1.6).

The enzymes may be co-expressed from different vectors, from one vector,or using a mixture of both techniques. When using different vectors, thevectors may have different selectable markers, and different origins ofreplication. When using only one vector, the genes can be expressed fromone or more promoters. If cloned under the regulation of one promoter(di- or multi-cistronic), the order in which the genes are cloned mayaffect the expression levels of the proteins. The protease may also beexpressed as a fusion protein, i.e. that the gene encoding the proteasehas been fused in frame to the gene encoding another protein. Thisprotein may be another enzyme or a functional domain from anotherenzyme.

Host Cells

The present invention also relates to recombinant host cells, comprisinga nucleic acid sequence of the invention, which are advantageously usedin the recombinant production of the polypeptides. A vector comprising anucleic acid sequence of the present invention is introduced into a hostcell so that the vector is maintained as a chromosomal integrant or as aself-replicating extra-chromosomal vector as described earlier. The term“host cell” encompasses any progeny of a parent cell that is notidentical to the parent cell due to mutations that occur duringreplication. The choice of a host cell will to a large extent dependupon the gene encoding the polypeptide and its source.

The host cell may be a unicellular microorganism, e.g., a prokaryote, ora non-unicellular microorganism, e.g., a eukaryote.

Useful unicellular cells are bacterial cells such as gram positivebacteria including, but not limited to, a Bacillus cell, or aStreptomyces cell, or cells of lactic acid bacteria; or gram negativebacteria such as E. coli and Pseudomonas sp. Lactic acid bacteriainclude, but are not limited to, species of the genera Lactococcus,Lactobacillus, Leuconostoc, Streptococcus, Pediococcus, andEnterococcus.

The introduction of a vector into a bacterial host cell may, forinstance, be effected by protoplast transformation (see, e.g., Chang andCohen, 1979, Molecular General Genetics 168: 111–115), using competentcells (see, e.g., Young and Spizizin, 1961, Journal of Bacteriology 81:823–829, or Dubnau and Davidoff-Abelson, 1971, Journal of MolecularBiology 56: 209–221), electroporation (see, e.g., Shigekawa and Dower,1988, Biotechniques 6: 742–751), or conjugation (see, e.g., Koehler andThorne, 1987, Journal of Bacteriology 169: 5771–5278).

The host cell may be a eukaryote, such as a non-human animal cell, aninsect cell, a plant cell, or a fungal cell.

In one particular embodiment, the host cell is a fungal cell. “Fungi” asused herein includes the phyla Ascomycota, Basidiomycota,Chytridiomycota, and Zygomycota (as defined by Hawksworth et al., In,Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CABInternational, University Press, Cambridge, UK) as well as the Oomycota(as cited in Hawksworth et al., 1995, supra, page 171) and allmitosporic fungi (Hawksworth et al., 1995, supra).

In another particular embodiment, the fungal host cell is a yeast cell.“Yeast” as used herein includes ascosporogenous yeast (Endomycetales),basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti(Blastomycetes). Since the classification of yeast may change in thefuture, for the purposes of this invention, yeast shall be defined asdescribed in Biology and Activities of Yeast (Skinner, F. A., Passmore,S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium SeriesNo. 9, 1980).

The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia cell.

The fungal host cell may be a filamentous fungal cell. “Filamentousfungi” include all filamentous forms of the subdivision Eumycota andOomycota (as defined by Hawksworth et al., 1995, supra). The filamentousfungi are characterized by a mycelial wall composed of chitin,cellulose, glucan, chitosan, mannan, and other complex polysaccharides.Vegetative growth is by hyphal elongation and carbon catabolism isobligately aerobic. In contrast, vegetative growth by yeasts such asSaccharomyces cerevisiae is by budding of a unicellular thallus andcarbon catabolism may be fermentative.

Examples of filamentous fungal host cells are cells of species of, butnot limited to, Acremonium, Aspergillus, Fusarium, Humicola, Mucor,Myceliophthora, Neurospora, Penicillium, Thielavia, Tolypocladium, orTrichoderma.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus host cells are described in EP 238 023 andYelton et al., 1984, Proceedings of the National Academy of Sciences USA81: 1470–1474. Suitable methods for transforming Fusarium species aredescribed by Malardier et al., 1989, Gene 78: 147–156 and WO 96/00787.Yeast may be transformed using the procedures described by Becker andGuarente, In Abelson, J. N. and Simon, M. I., editors, Guide to YeastGenetics and Molecular Biology, Methods in Enzymology, Volume 194, pp182–187, Academic Press, Inc., New York; Ito et al., 1983, Journal ofBacteriology 153: 163; and Hinnen et al., 1978, Proceedings of theNational Academy of Sciences USA 75: 1920.

Methods of Production

The present invention also relates to methods for producing apolypeptide of the present invention comprising (a) cultivating astrain, which in its wild-type form is capable of producing thepolypeptide; and (b) recovering the polypeptide. Preferably, the strainis of the genus Nocardiopsis, more preferably Nocardiopsis prasina,Nocardiopsis antarctica, Nocardiopsis dassonvillei or Nocardiopsis alba,most preferably Nocardiopsis alba DSM 15647, or Nocardiopsisdassonvillei subsp. dassonvillei DSM 43235.

The present invention also relates to methods for producing apolypeptide of the present invention comprising (a) cultivating a hostcell under conditions conducive for production of the polypeptide; and(b) recovering the polypeptide.

The present invention also relates to methods for producing apolypeptide of the present invention comprising (a) cultivating a hostcell under conditions conducive for production of the polypeptide,wherein the host cell comprises a mutant nucleic acid sequencecomprising at least one mutation in nucleotides 502–1065, or 1–1065, ofSEQ ID NO: 1, or in nucleotides 1–1143, 88–1143, preferably 568–1143 ofSEQ ID NO: 5, in which the mutant nucleic acid sequence encodes apolypeptide which (i) consists of amino acids 1 to 188, or −167 to 188,of SEQ ID NO: 2, or amino acids −160 to 192, preferably 1–192, of SEQ IDNO: 6, or (ii) is a variant of any of the sequences of (i), wherein thevariant comprises a substitution, deletion, and/or insertion of one ormore amino acids, or (iii) is an allelic variant of any of the sequencesof (i), or (iv) is a fragment of any of the sequences of (i).

In the production methods of the present invention, the cells arecultivated in a nutrient medium suitable for production of thepolypeptide using methods known in the art. For example, the cell may becultivated by shake flask cultivation, small-scale or large-scalefermentation (including continuous, batch, fed-batch, or solid statefermentations) in laboratory or industrial fermentors performed in asuitable medium and under conditions allowing the polypeptide to beexpressed and/or isolated. The cultivation takes place in a suitablenutrient medium comprising carbon and nitrogen sources and inorganicsalts, using procedures known in the art. Suitable media are availablefrom commercial suppliers or may be prepared according to publishedcompositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted, it can be recovered from cell lysates.

The polypeptides may be detected using methods known in the art that arespecific for the polypeptides. These detection methods may include useof specific antibodies, formation of a product, or disappearance of asubstrate. For example, a protease assay may be used to determine theactivity of the polypeptide as described herein.

The resulting polypeptide may be recovered by methods known in the art.For example, the polypeptide may be recovered from the nutrient mediumby conventional procedures including, but not limited to,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The polypeptides of the present invention may be purified by a varietyof procedures known in the art including, but not limited to,chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing), differential solubility (e.g.,ammonium sulphate precipitation), SDS-PAGE, or extraction (see, e.g.,Protein Purification, J.-C. Janson and Lars Ryden, editors VCHPublishers, New York, 1989).

Plants

The present invention also relates to a transgenic plant, plant part, orplant cell which has been transformed with a nucleic acid sequenceencoding a polypeptide having protease activity of the present inventionso as to express and produce the polypeptide in recoverable quantities.The polypeptide may be recovered from the plant or plant part.Alternatively, the plant or plant part containing the recombinantpolypeptide may be used as such for improving the quality of a food orfeed, e.g., improving nutritional value, palatability, and rheologicalproperties, or to destroy an antinutritive factor.

In a particular embodiment, the polypeptide is targeted to the endospermstorage vacuoles in seeds. This can be obtained by synthesizing it as aprecursor with a suitable signal peptide, see Horvath et al in PNAS,Feb. 15, 2000, vol. 97, no. 4, p. 1914–1919.

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous(a monocot) or engineered variants thereof. Examples of monocot plantsare grasses, such as meadow grass (blue grass, Poa), forage grass suchas Festuca, Lolium, temperate grass, such as Agrostis, and cereals,e.g., wheat, oats, rye, barley, rice, sorghum, triticale (stabilizedhybrid of wheat (Triticum) and rye (Secale), and maize (corn). Examplesof dicot plants are tobacco, legumes, such as lupins, potato, sugarbeet, pea, bean and soybean, and cruciferous plants (familyBrassicaceae), such as sunflower (Helianthus), cotton (Gossypium),cauliflower, rape seed, and the closely related model organismArabidopsis thaliana. Low-phytate plants as described e.g. in U.S. Pat.Nos. 5,689,054 and 6,111,168 are examples of engineered plants.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers, as well as the individual tissues comprising these parts,e.g. epidermis, mesophyll, parenchyma, vascular tissues, meristems. Alsospecific plant cell compartments, such as chloroplast, apoplast,mitochondria, vacuole, peroxisomes, and cytoplasm are considered to be aplant part. Furthermore, any plant cell, whatever the tissue origin, isconsidered to be a plant part. Likewise, plant parts such as specifictissues and cells isolated to facilitate the utilisation of theinvention are also considered plant parts, e.g. embryos, endosperms,aleurone and seed coats.

Also included within the scope of the present invention are the progenyof such plants, plant parts and plant cells.

The transgenic plant or plant cell expressing a polypeptide of thepresent invention may be constructed in accordance with methods known inthe art. Briefly, the plant or plant cell is constructed byincorporating one or more expression constructs encoding a polypeptideof the present invention into the plant host genome and propagating theresulting modified plant or plant cell into a transgenic plant or plantcell.

Conveniently, the expression construct is a nucleic acid construct whichcomprises a nucleic acid sequence encoding a polypeptide of the presentinvention operably linked with appropriate regulatory sequences requiredfor expression of the nucleic acid sequence in the plant or plant partof choice. Furthermore, the expression construct may comprise aselectable marker useful for identifying host cells into which theexpression construct has been integrated and DNA sequences necessary forintroduction of the construct into the plant in question (the latterdepends on the DNA introduction method to be used).

The choice of regulatory sequences, such as promoter and terminatorsequences and optionally signal or transit sequences are determined, forexample, on the basis of when, where, and how the polypeptide is desiredto be expressed. For instance, the expression of the gene encoding apolypeptide of the present invention may be constitutive or inducible,or may be developmental, stage or tissue specific, and the gene productmay be targeted to a specific tissue or plant part such as seeds orleaves. Regulatory sequences are, for example, described by Tague etal., 1988, Plant Physiology 86: 506.

For constitutive expression, the following promoters may be used: The35S-CaMV promoter (Franck et al., 1980, Cell 21: 285–294), the maizeubiquitin 1 (Christensen A H, Sharrock R A and Quail 1992. Maizepolyubiquitin genes: structure, thermal perturbation of expression andtranscript splicing, and promoter activity following transfer toprotoplasts by electroporation), or the rice actin 1 promoter (Plant Mo.Biol. 18, 675–689.; Zhang W, McElroy D. and Wu R 1991, Analysis of riceAct1 5′ region activity in transgenic rice plants. Plant Cell 3,1155–1165). Organ-specific promoters may be, for example, a promoterfrom storage sink tissues such as seeds, potato tubers, and fruits(Edwards & Coruzzi, 1990, Ann. Rev. Genet. 24: 275–303), or frommetabolic sink tissues such as meristems (Ito et al., 1994, Plant Mol.Biol. 24: 863–878), a seed specific promoter such as the glutelin,prolamin, globulin, or albumin promoter from rice (Wu et al., 1998,Plant and Cell Physiology 39: 885–889), a Vicia faba promoter from thelegumin B4 and the unknown seed protein gene from Vicia faba (Conrad etal., 1998, Journal of Plant Physiology 152: 708–711), a promoter from aseed oil body protein (Chen et al., 1998, Plant and Cell Physiology 39:935–941), the storage protein napA promoter from Brassica napus, or anyother seed specific promoter known in the art, e.g., as described in WO91/14772. Furthermore, the promoter may be a leaf specific promoter suchas the rbcs promoter from rice or tomato (Kyozuka et al., 1993, PlantPhysiology 102: 991–1000, the chlorella virus adenine methyltransferasegene promoter (Mitra and Higgins, 1994, Plant Molecular Biology 26:85–93), or the aldP gene promoter from rice (Kagaya et al., 1995,Molecular and General Genetics 248: 668–674), or a wound induciblepromoter such as the potato pin2 promoter (Xu et al., 1993, PlantMolecular Biology 22: 573–588). Likewise, the promoter may be inducibleby abiotic treatments such as temperature, drought or alterations insalinity or inducible by exogenously applied substances that activatethe promoter, e.g. ethanol, oestrogens, plant hormones like ethylene,abscisic acid, gibberellic acid, and/or heavy metals.

A promoter enhancer element may also be used to achieve higherexpression of the protease in the plant. For instance, the promoterenhancer element may be an intron which is placed between the promoterand the nucleotide sequence encoding a polypeptide of the presentinvention. For instance, Xu et al., 1993, supra disclose the use of thefirst intron of the rice actin 1 gene to enhance expression.

Still further, the codon usage may be optimized for the plant species inquestion to improve expression (see Horvath et al referred to above).

The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

The nucleic acid construct is incorporated into the plant genomeaccording to conventional techniques known in the art, includingAgrobacterium-mediated transformation, virus-mediated transformation,microinjection, particle bombardment, biolistic transformation, andelectroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990,Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).

Presently, Agrobacterium tumefaciens-mediated gene transfer is themethod of choice for generating transgenic dicots (for a review, seeHooykas and Schilperoort, 1992, Plant Molecular Biology 19: 15–38), andit can also be used for transforming monocots, although othertransformation methods are generally preferred for these plants.Presently, the method of choice for generating transgenic monocots,supplementing the Agrobacterium approach, is particle bombardment(microscopic gold or tungsten particles coated with the transformingDNA) of embryonic calli or developing embryos (Christou, 1992, PlantJournal 2: 275–281; Shimamoto, 1994, Current Opinion Biotechnology 5:158–162; Vasil et al., 1992, Bio/Technology 10: 667–674). An alternativemethod for transformation of monocots is based on protoplasttransformation as described by Omirulleh et al., 1993, Plant MolecularBiology 21: 415–428.

Following transformation, the transformants having incorporated thereinthe expression construct are selected and regenerated into whole plantsaccording to methods well-known in the art.

The present invention also relates to methods for producing apolypeptide of the present invention comprising (a) cultivating atransgenic plant or a plant cell comprising a nucleic acid sequenceencoding a polypeptide having protease activity of the present inventionunder conditions conducive for production of the polypeptide; and (b)recovering the polypeptide.

Animals

The present invention also relates to a transgenic, non-human animal andproducts or elements thereof, examples of which are body fluids such asmilk and blood, organs, flesh, and animal cells. Techniques forexpressing proteins, e.g. in mammalian cells, are known in the art, seee.g. the handbook Protein Expression: A Practical Approach, Higgins andHames (eds), Oxford University Press (1999), and the three otherhandbooks in this series relating to Gene Transcription, RNA processing,and Post-translational Processing. Generally speaking, to prepare atransgenic animal, selected cells of a selected animal are transformedwith a nucleic acid sequence encoding a polypeptide having proteaseactivity of the present invention so as to express and produce thepolypeptide. The polypeptide may be recovered from the animal, e.g. fromthe milk of female animals, or the polypeptide may be expressed to thebenefit of the animal itself, e.g. to assist the animal's digestion.Examples of animals are mentioned below in the section headed AnimalFeed.

To produce a transgenic animal with a view to recovering the proteasefrom the milk of the animal, a gene encoding the protease may beinserted into the fertilized eggs of an animal in question, e.g. by useof a transgene expression vector which comprises a suitable milk proteinpromoter, and the gene encoding the protease. The transgene expressionvector is microinjected into fertilized eggs, and preferably permanentlyintegrated into the chromosome. Once the egg begins to grow and divide,the potential embryo is implanted into a surrogate mother, and animalscarrying the transgene are identified. The resulting animal can then bemultiplied by conventional breeding. The polypeptide may be purifiedfrom the animal's milk, see e.g. Meade, H. M. et al (1999): Expressionof recombinant proteins in the milk of transgenic animals, Geneexpression systems: Using nature for the art of expression. J. M.Fernandez and J. P. Hoeffler (eds.), Academic Press.

In the alternative, in order to produce a transgenic non-human animalthat carries in the genome of its somatic and/or germ cells a nucleicacid sequence including a heterologous transgene construct including atransgene encoding the protease, the transgene may be operably linked toa first regulatory sequence for salivary gland specific expression ofthe protease, as disclosed in WO 2000/064247.

Compositions

In a still further aspect, the present invention relates to compositionscomprising a polypeptide of the present invention.

The polypeptide compositions may be prepared in accordance with methodsknown in the art and may be in the form of a liquid or a drycomposition. For instance, the polypeptide composition may be in theform of a granulate or a microgranulate. The polypeptide to be includedin the composition may be stabilized in accordance with methods known inthe art.

Examples are given below of preferred uses of the polypeptides orpolypeptide compositions of the invention.

Animal Feed

The present invention is also directed to methods for using thepolypeptides of the invention in animal feed, as well as to feedcompositions and feed additives comprising the polypeptides of theinvention.

The term animal includes all animals, including human beings. Examplesof animals are non-ruminants, and ruminants. Ruminant animals include,for example, animals such as sheep, goats, horses, and cattle, e.g. beefcattle, cows, and young calves. In a particular embodiment, the animalis a non-ruminant animal. Non-ruminant animals include mono-gastricanimals, e.g. pigs or swine (including, but not limited to, piglets,growing pigs, and sows); poultry such as turkeys, ducks and chicken(including but not limited to broiler chicks, layers); young calves; andfish (including but not limited to salmon, trout, tilapia, catfish andcarps; and crustaceans (including but not limited to shrimps andprawns).

The term feed or feed composition means any compound, preparation,mixture, or composition suitable for, or intended for intake by ananimal.

In the use according to the invention the protease can be fed to theanimal before, after, or simultaneously with the diet. The latter ispreferred.

In a particular embodiment, the protease, in the form in which it isadded to the feed, or when being included in a feed additive, iswell-defined. Well-defined means that the protease preparation is atleast 50% pure as determined by Size-exclusion chromatography (seeExample 12 of WO 01/58275). In other particular embodiments the proteasepreparation is at least 60, 70, 80, 85, 88, 90, 92, 94, or at least 95%pure as determined by this method.

A well-defined protease preparation is advantageous. For instance, it ismuch easier to dose correctly to the feed a protease that is essentiallyfree from interfering or contaminating other proteases. The term dosecorrectly refers in particular to the objective of obtaining consistentand constant results, and the capability of optimising dosage based uponthe desired effect.

For the use in animal feed, however, the protease need not be that pure;it may e.g. include other enzymes, in which case it could be termed aprotease preparation.

The protease preparation can be (a) added directly to the feed (or useddirectly in a treatment process of proteins), or (b) it can be used inthe production of one or more intermediate compositions such as feedadditives or premixes that is subsequently added to the feed (or used ina treatment process). The degree of purity described above refers to thepurity of the original protease preparation, whether used according to(a) or (b) above.

Protease preparations with purities of this order of magnitude are inparticular obtainable using recombinant methods of production, whereasthey are not so easily obtained and also subject to a much higherbatch-to-batch variation when the protease is produced by traditionalfermentation methods.

Such protease preparation may of course be mixed with other enzymes.

In a particular embodiment, the protease for use according to theinvention is capable of solubilising proteins. A suitable assay fordetermining solubilised protein is disclosed in Example 5.

The protein may be an animal protein, such as meat and bone meal, and/orfish meal; or it may be a vegetable protein. The term vegetable proteinsas used herein refers to any compound, composition, preparation ormixture that includes at least one protein derived from or originatingfrom a vegetable, including modified proteins and protein-derivatives.In particular embodiments, the protein content of the vegetable proteinsis at least 10, 20, 30, 40, 50, or 60% (w/w).

Vegetable proteins may be derived from vegetable protein sources, suchas legumes and cereals, for example materials from plants of thefamilies Fabaceae (Leguminosae), Cruciferaceae, Chenopodiaceae, andPoaceae, such as soy bean meal, lupin meal and rapeseed meal.

In a particular embodiment, the vegetable protein source is materialfrom one or more plants of the family Fabaceae, e.g. soybean, lupine,pea, or bean.

In another particular embodiment, the vegetable protein source ismaterial from one or more plants of the family Chenopodiaceae, e.g.beet, sugar beet, spinach or quinoa.

Other examples of vegetable protein sources are rapeseed, sunflowerseed, cotton seed, and cabbage.

Soybean is a preferred vegetable protein source.

Other examples of vegetable protein sources are cereals such as barley,wheat, rye, oat, maize (corn), rice, triticale, and sorghum.

The treatment according to the invention of proteins with at least oneprotease of the invention results in an increased solubilisation ofproteins.

The following are examples of % solubilised protein obtainable using theproteases of the invention in a monogastric in vitro model: At least101%, or at least 102%, 103%, 104%, 105%, 106%, or at least 107%,relative to a blank. The percentage of solubilised protein is determinedusing the monogastric in vitro model of Example 5. The termsolubilisation of proteins basically means bringing protein(s) intosolution. Such solubilisation may be due to protease-mediated release ofprotein from other components of the usually complex naturalcompositions such as feed.

In a further particular embodiment, the protease for use according tothe invention is capable of increasing the amount of digestibleproteins. The following are examples of % digested or digestible proteinobtainable using the proteases of the invention in a monogastric invitro model: At least 101%, or at least 102%, relative to a blank. Thepercentage of digested or digestible protein is determined using the invitro model of Example 5.

In a still further particular embodiment, the protease for use accordingto the invention is capable of increasing the Degree of Hydrolysis (DH)of proteins. In a particular embodiment, the degree of hydrolysis is atleast 101%, 102%, 103%, 104%, or at least 105%, relative to a blank. Thedegree of hydrolysis is determined using the in vitro model of Example5.

In a particular embodiment of a (pre-) treatment process of theinvention, the protease(s) in question is affecting (or acting on, orexerting its solubilising influence on) the proteins or protein sources.To achieve this, the protein or protein source is typically suspended ina solvent, e.g. an aqueous solvent such as water, and the pH andtemperature values are adjusted paying due regard to the characteristicsof the enzyme in question. For example, the treatment may take place ata pH-value at which the activity of the actual protease is at least atleast 40%, 50%, 60%, 70%, 80% or at least 90%. Likewise, for example,the treatment may take place at a temperature at which the activity ofthe actual protease is at least 40%, 50%, 60%, 70%, 80% or at least 90%.The above percentage activity indications are relative to the maximumactivities. The enzymatic reaction is continued until the desired resultis achieved, following which it may or may not be stopped byinactivating the enzyme, e.g. by a heat-treatment step.

In another particular embodiment of a treatment process of theinvention, the protease action is sustained, meaning e.g. that theprotease is added to the proteins or protein sources, but itssolubilising influence is so to speak not switched on until later whendesired, once suitable solubilising conditions are established, or onceany enzyme inhibitors are inactivated, or whatever other means couldhave been applied to postpone the action of the enzyme.

In one embodiment the treatment is a pre-treatment of animal feed orproteins for use in animal feed.

The term improving the nutritional value of an animal feed meansimproving the availability and/or digestibility of the proteins, therebyleading to increased protein extraction from the diet components, higherprotein yields, increased protein degradation and/or improved proteinutilisation. The nutritional value of the feed is therefore increased,and the animal performance such as growth rate and/or weight gain and/orfeed conversion ratio (i.e. the weight of ingested feed relative toweight gain) of the animal is/are improved.

In a particular embodiment the feed conversion ratio is increased by atleast 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or at least10%. In a furtherparticular embodiment the weight gain is increased by at least 2%, 3%,4%, 5%, 6%, 7%, 8%, 9%, 10% or at least 11%. These figures are relativeto control experiments with no protease addition.

The feed conversion ratio (FCR) and the weight gain may be calculated asdescribed in EEC (1986): Directive de la Commission du 9 avril 1986fixant la méthode de calcul de la valeur énérgetique des alimentscomposés destinés à la volaille. Journal Officiel des CommunautésEuropéennes, L1 30, 53–54.

The protease can be added to the feed in any form, be it as a relativelypure protease, or in admixture with other components intended foraddition to animal feed, i.e. in the form of animal feed additives, suchas the so-called pre-mixes for animal feed.

In a further aspect the present invention relates to compositions foruse in animal feed, such as animal feed, and animal feed additives, e.g.premixes.

Apart from the protease of the invention, the animal feed additives ofthe invention contain at least one fat-soluble vitamin, and/or at leastone water soluble vitamin, and/or at least one trace mineral. The feedadditive may also contain at least one macro mineral.

Further, optional, feed-additive ingredients are colouring agents, e.g.carotenoids such as beta-carotene, astaxanthin, and lutein; aromacompounds; stabilisers; antimicrobial peptides; polyunsaturated fattyacids; reactive oxygen generating species; and/or at least one otherenzyme selected from amongst amylase such as, for example, amylase suchas alpha-amylase (EC 3.2.1.1), phytase (EC 3.1.3.8 or 3.1.3.26);xylanase (EC 3.2.1.8); galactanase (EC 3.2.1.89); alpha-galactosidase(EC 3.2.1.22); protease (EC 3.4.−.−), phospholipase A1 (EC 3.1.1.32);phospholipase A2 (EC 3.1.1.4); lysophospholipase (EC 3.1.1.5);phospholipase C (3.1.4.3); phospholipase D (EC 3.1.4.4); and/orbeta-glucanase (EC 3.2.1.4 or EC 3.2.1.6).

In a particular embodiment these other enzymes are well-defined (asdefined above for protease preparations).

Examples of antimicrobial peptides (AMP's) are CAP18, Leucocin A,Tritrpticin, Protegrin-1, Thanatin, Defensin, Lactoferrin,Lactoferricin, and Ovispirin such as Novispirin (Robert Lehrer, 2000),Plectasins, and Statins, including the compounds and polypeptidesdisclosed in WO 03/044049 and WO 03/048148, as well as variants orfragments of the above that retain antimicrobial activity.

Examples of antifungal polypeptides (AFP's) are the Aspergillusgiganteus, and Aspergillus niger peptides, as well as variants andfragments thereof which retain antifungal activity, as disclosed in WO94/01459 and WO 02/090384.

Examples of polyunsaturated fatty acids are C18, C20 and C22polyunsaturated fatty acids, such as arachidonic acid, docosohexaenoicacid, eicosapentaenoic acid and gamma-linoleic acid.

Examples of reactive oxygen generating species are chemicals such asperborate, persulphate, or percarbonate; and enzymes such as an oxidase,an oxygenase or a syntethase.

Usally fat- and water-soluble vitamins, as well as trace minerals formpart of a so-called premix intended for addition to the feed, whereasmacro minerals are usually separately added to the feed. A premixenriched with a protease of the invention, is an example of an animalfeed additive of the invention.

In a particular embodiment, the animal feed additive of the invention isintended for being included (or prescribed as having to be included) inanimal diets or feed at levels of 0.01 to 10.0%; more particularly 0.05to 5.0%; or 0.2 to 1.0% (% meaning g additive per 100 g feed). This isso in particular for premixes.

The following are non-exclusive lists of examples of these components:

Examples of fat-soluble vitamins are vitamin A, vitamin D3, vitamin E,and vitamin K, e.g. vitamin K3.

Examples of water-soluble vitamins are vitamin B12, biotin and choline,vitamin B1, vitamin B2, vitamin B6, niacin, folic acid andpanthothenate, e.g. Ca-D-panthothenate.

Examples of trace minerals are manganese, zinc, iron, copper, iodine,selenium, and cobalt.

Examples of macro minerals are calcium, phosphorus and sodium.

The nutritional requirements of these components (exemplified withpoultry and piglets/pigs) are listed in Table A of WO 01/58275.Nutritional requirement means that these components should be providedin the diet in the concentrations indicated.

In the alternative, the animal feed additive of the invention comprisesat least one of the individual components specified in Table A of WO01/58275. At least one means either of, one or more of, one, or two, orthree, or four and so forth up to all thirteen, or up to all fifteenindividual components. More specifically, this at least one individualcomponent is included in the additive of the invention in such an amountas to provide an in-feed-concentration within the range indicated incolumn four, or column five, or column six of Table A.

The present invention also relates to animal feed compositions. Animalfeed compositions or diets have a relatively high content of protein.Poultry and pig diets can be characterised as indicated in Table B of WO01/58275, columns 2–3. Fish diets can be characterised as indicated incolumn 4 of this Table B. Furthermore such fish diets usually have acrude fat content of 200–310 g/kg. WO 01/58275 corresponds to U.S. Ser.No. 09/77334 which is hereby incorporated by reference.

An animal feed composition according to the invention has a crudeprotein content of 50–800 g/kg, and furthermore comprises at least oneprotease as claimed herein.

Furthermore, or in the alternative (to the crude protein contentindicated above), the animal feed composition of the invention has acontent of metabolisable energy of 10–30 MJ/kg; and/or a content ofcalcium of 0.1–200 g/kg; and/or a content of available phosphorus of0.1–200 g/kg; and/or a content of methionine of 0.1–100 g/kg; and/or acontent of methionine plus cysteine of 0.1–150 g/kg; and/or a content oflysine of 0.5–50 g/kg.

In particular embodiments, the content of metabolisable energy, crudeprotein, calcium, phosphorus, methionine, methionine plus cysteine,and/or lysine is within any one of ranges 2, 3, 4 or 5 in Table B of WO01/58275 (R. 2–5).

Crude protein is calculated as nitrogen (N) multiplied by a factor 6.25,i.e. Crude protein (g/kg)=N (g/kg)×6.25. The nitrogen content isdetermined by the Kjeldahl method (A.O.A.C., 1984, Official Methods ofAnalysis 14th ed., Association of Official Analytical Chemists,Washington D.C.).

Metabolisable energy can be calculated on the basis of the NRCpublication Nutrient requirements in swine, ninth revised edition 1988,subcommittee on swine nutrition, committee on animal nutrition, board ofagriculture, national research council. National Academy Press,Washington, D.C., pp. 2–6, and the European Table of Energy Values forPoultry Feed-stuffs, Spelderholt centre for poultry research andextension, 7361 DA Beekbergen, The Netherlands. Grafisch bedrijf Ponsen& looijen bv, Wageningen. ISBN 90-71463-12-5.

The dietary content of calcium, available phosphorus and amino acids incomplete animal diets is calculated on the basis of feed tables such asVeevoedertabel 1997, gegevens over chemische samenstelling,verteerbaarheid en voederwaarde van voedermiddelen, CentralVeevoederbureau, Runderweg 6, 8219 pk Lelystad. ISBN 90-72839-13-7.

In a particular embodiment, the animal feed composition of the inventioncontains at least one protein or protein source as defined above. It mayalso contain animal protein, such as Meat and Bone Meal, and/or FishMeal, typically in an amount of 0–25%.

In still further particular embodiments, the animal feed composition ofthe invention contains 0–80% maize; and/or 0–80% sorghum; and/or 0–70%wheat; and/or 0–70% Barley; and/or 0–30% oats; and/or 0–40% soybeanmeal; and/or 0–25% fish meal; 0–25% meat and bone meal; and/or 0–20%whey.

Animal diets can e.g. be manufactured as mash feed (non pelleted) orpelleted feed. Typically, the milled feed-stuffs are mixed andsufficient amounts of essential vitamins and minerals are addedaccording to the specifications for the species in question. Enzymes canbe added as solid or liquid enzyme formulations. For example, a solidenzyme formulation is typically added before or during the mixing step;and a liquid enzyme preparation is typically added after the pelletingstep. The enzyme may also be incorporated in a feed additive or premix.

The final enzyme concentration in the diet is within the range of0.01–200 mg enzyme protein per kg diet, for example in the range of0.5–25 mg enzyme protein per kg animal diet.

The protease should of course be applied in an effective amount, i.e. inan amount adequate for improving solubilisation and/or improvingnutritional value of feed. It is at present contemplated that the enzymeis administered in one or more of the following amounts (dosage ranges):0.01–200; 0.01–100; 0.5–100; 1–50; 5–100; 10–100; 0.05–50; or 0.10–10—all these ranges being in mg protease enzyme protein per kg feed (ppm).

For determining mg enzyme protein per kg feed, the protease is purifiedfrom the feed composition, and the specific activity of the purifiedprotease is determined using a relevant assay (see under proteaseactivity, substrates, and assays). The protease activity of the feedcomposition as such is also determined using the same assay, and on thebasis of these two determinations, the dosage in mg enzyme protein perkg feed is calculated.

The same principles apply for determining mg enzyme protein in feedadditives. Of course, if a sample is available of the protease used forpreparing the feed additive or the feed, the specific activity isdetermined from this sample (no need to purify the protease from thefeed composition or the additive).

The present invention is further described by the following exampleswhich should not be construed as limiting the scope of the invention.

Detergent Compositions

The protease of the invention may be added to and thus become acomponent of a detergent composition.

The detergent composition of the invention may for example be formulatedas a hand or machine laundry detergent composition including a laundryadditive composition suitable for pre-treatment of stained fabrics and arinse added fabric softener composition, or be formulated as a detergentcomposition for use in general household hard surface cleaningoperations, or be formulated for hand or machine dishwashing operations.

In a specific aspect, the invention provides a detergent additivecomprising the protease of the invention. The detergent additive as wellas the detergent composition may comprise one or more other enzymes suchas another protease, such as alkaline proteases from Bacillus, a lipase,a cutinase, an amylase, a carbohydrase, a cellulase, a pectinase, amannanase, an arabinase, a galactanase, a xylanase, an oxidase, e.g., alaccase, and/or a peroxidase.

In general the properties of the chosen enzyme(s) should be compatiblewith the selected detergent, (i.e. pH-optimum, compatibility with otherenzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) shouldbe present in effective amounts.

Suitable lipases include those of bacterial or fungal origin. Chemicallymodified or protein engineered mutants are included. Examples of usefullipases include lipases from Humicola (synonym Thermomyces), e.g. fromH. lanuginosa (T. lanuginosus) as described in EP 258068 and EP 305216or from H. insolens as described in WO 96/13580, a Pseudomonas lipase,e.g. from P. alcaligenes or P. pseudoalcaligenes (EP 218272), P. cepacia(EP 331376), P. stutzeri (GB 1,372,034), P. fluorescens, Pseudomonas sp.strain SD 705 (WO 95/06720 and WO 96/27002), P. wisconsinensis (WO96/12012), a Bacillus lipase, e.g. from B. subtilis (Dartois et al.(1993), Biochemica et Biophysica Acta, 1131, 253–360), B.stearothermophilus (JP 64/744992) or B. pumilus (WO 91/16422). Otherexamples are lipase variants such as those described in WO 92/05249, WO94/01541, EP 407225, EP 260105, WO 95/35381, WO 96/00292, WO 95/30744,WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079 and WO 97/07202.Preferred commercially available lipase enzymes include Lipolase™ andLipolase Ultra™0 (Novozymes A/S).

Suitable amylases (alpha- and/or beta-) include those of bacterial orfungal origin. Chemically modified or protein engineered mutants areincluded. Amylases include, for example, alpha-amylases obtained fromBacillus, e.g. a special strain of B. licheniformis, described in moredetail in GB 1,296,839. Examples of useful amylases are the variantsdescribed in WO 94/02597, WO 94/18314, WO 95/26397, WO 96/23873, WO97/43424, WO 00/60060, and WO 01/66712, especially the variants withsubstitutions in one or more of the following positions: 15, 23, 105,106, 124, 128, 133, 154, 156, 181, 188, 190, 197, 202, 208, 209, 243,264, 304, 305, 391, 408, and 444. Commercially available amylases areNatalase™, SupraMyl™, Stainzyme™, Duramyl™, Termamyl™, Fungamyl™ andBAN™ (Novozymes A/S), Rapidase™ and Purastar™ (from GenencorInternational Inc.).

Suitable cellulases include those of bacterial or fungal origin.Chemically modified or protein engineered mutants are included. Suitablecellulases include cellulases from the genera Bacillus, Pseudomonas,Humicola, Fusarium, Thielavia, Acremonium, e.g. the fungal cellulasesproduced from Humicola insolens, Myceliophthora thermophila and Fusariumoxysporum disclosed in U.S. Pat. Nos. 4,435,307, 5,648,263, 5,691,178,5,776,757 and WO 89/09259. Especially suitable cellulases are thealkaline or neutral cellulases having colour care benefits. Examples ofsuch cellulases are cellulases described in EP 0 495257, EP 531372, WO96/11262, WO 96/29397, WO 98/08940. Other examples are cellulasevariants such as those described in WO 94/07998, EP 0 531 315, U.S. PatNos. 5,457,046, 5,686,593, 5,763,254, WO 95/24471, WO 98/12307 and WO99/01544. Commercially available cellulases include Celluzyme™, andCarezyme™ (Novozymes A/S), Clazinase™, and Puradax HA™ (GenencorInternational Inc.), and KAC-500(B)™ (Kao Corporation).

Suitable peroxidases/oxidases include those of plant, bacterial orfungal origin. Chemically modified or protein engineered mutants areincluded. Examples of useful peroxidases include peroxidases fromCoprinus, e.g. from C. cinereus, and variants thereof as those describedin WO 93/24618, WO 95/10602, and WO 98/15257. Commercially availableperoxidases include Guardzyme™ (Novozymes).

The detergent enzyme(s) may be included in a detergent composition byadding separate additives containing one or more enzymes, or by adding acombined additive comprising all of these enzymes. A detergent additiveof the invention, i.e. a separate additive or a combined additive, canbe formulated e.g. as a granulate, a liquid, a slurry, etc. Preferreddetergent additive formulations are granulates, in particularnon-dusting granulates, liquids, in particular stabilized liquids, orslurries.

Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat.Nos. 4,106,991 and 4,661,452 and may optionally be coated by methodsknown in the art. Examples of waxy coating materials are poly(ethyleneoxide) products (polyethyleneglycol, PEG) with mean molar weights of1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethyleneoxide units; ethoxylated fatty alcohols in which the alcohol containsfrom 12 to 20 carbon atoms and in which there are 15 to 80 ethyleneoxide units; fatty alcohols; fatty acids; and mono- and di- andtriglycerides of fatty acids. Examples of film-forming coating materialssuitable for application by fluid bed techniques are given in GB1483591. Liquid enzyme preparations may, for instance, be stabilized byadding a polyol such as propylene glycol, a sugar or sugar alcohol,lactic acid or boric acid according to established methods. Protectedenzymes may be prepared according to the method disclosed in EP 238216.

The detergent composition of the invention may be in any convenientform, e.g., a bar, a tablet, a powder, a granule, a paste or a liquid. Aliquid detergent may be aqueous, typically containing up to 70% waterand 0–30% organic solvent, or non-aqueous.

The detergent composition comprises one or more surfactants, which maybe non-ionic including semi-polar and/or anionic and/or cationic and/orzwitterionic. The surfactants are typically present at a level of from0.1% to 60% by weight.

When included therein the detergent will usually contain from about 1%to about 40% of an anionic surfactant such as linearalkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fattyalcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate,alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid orsoap.

When included therein the detergent will usually contain from about 0.2%to about 40% of a non-ionic surfactant such as alcohol ethoxylate,nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamineoxide,ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide,polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl derivatives ofglucosamine (“glucamides”).

The detergent may contain 0–65% of a detergent builder or complexingagent such as zeolite, diphosphate, triphosphate, phosphonate,carbonate, citrate, nitrilotriacetic acid, ethylenediaminetetraaceticacid, diethylenetriaminepentaacetic acid, alkyl- or alkenylsuccinicacid, soluble silicates or layered silicates (e.g. SKS-6 from Hoechst).

The detergent may comprise one or more polymers. Examples arecarboxymethylcellulose, poly(vinylpyrrolidone), poly (ethylene glycol),poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole),polycarboxylates such as polyacrylates, maleic/acrylic acid copolymersand lauryl methacrylate/acrylic acid copolymers.

The detergent may contain a bleaching system which may comprise a H₂O₂source such as perborate or percarbonate which may be combined with aperacid-forming bleach activator such as tetraacetylethylenediamine ornonanoyloxybenzenesulfonate. Alternatively, the bleaching system maycomprise peroxyacids of e.g. the amide, imide, or sulfone type.

The enzyme(s) of the detergent composition of the invention may bestabilized using conventional stabilizing agents, e.g., a polyol such aspropylene glycol or glycerol, a sugar or sugar alcohol, lactic acid,boric acid, or a boric acid derivative, e.g., an aromatic borate ester,or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid,and the composition may be formulated as described in e.g. WO 92/19709and WO 92/19708.

The detergent may also contain other conventional detergent ingredientssuch as e.g. fabric conditioners including clays, foam boosters, sudssuppressors, anti-corrosion agents, soil-suspending agents, anti-soilredeposition agents, dyes, bactericides, optical brighteners,hydrotropes, tarnish inhibitors, or perfumes.

It is at present contemplated that in the detergent compositions anyenzyme, in particular the enzyme of the invention, may be added in anamount corresponding to 0.01–100 mg of enzyme protein per liter of washliqour, preferably 0.05–5 mg of enzyme protein per liter of wash liqour,in particular 0.1–1 mg of enzyme protein per liter of wash liqour.

The enzyme of the invention may additionally be incorporated in thedetergent formulations disclosed in WO 97/07202.

Deposit of Biological Material

The following biological material, isolated from a soil sample collectedin Denmark in 2001, has been deposited under the terms of the BudapestTreaty with the Deutsche Sammlung von Mikroorganismen und ZelikulturenGmbH, Mascheroder Weg 1b, D-38124 Braunschweig, and given the followingaccession number:

Deposit Accession Number Date of Deposit Nocardiopsis alba DSM 15647 May30, 2003

The strain has been deposited under conditions that assure that accessto the culture will be available during the pendency of the patentapplications to one determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 C.F.R. § 1.14 and 35 U.S.C. §122. The deposit represents a substantially pure culture of thedeposited strain. The deposit is available as required by foreign patentlaws in countries wherein counterparts of these applications, or theirprogeny are filed. However, it should be understood that theavailability of a deposit does not constitute a license to practice theinvention in derogation of patent rights granted by governmental action.

The invention described and claimed herein is not to be limited in scopeby the specific embodiments herein disclosed, since these embodimentsare intended as illustrations of several aspects of the invention. Anyequivalent embodiments are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

EXAMPLES Example 1 Cloning and Expression of the Protease Derived fromNocardiopsis alba DSM 15647

Reagents and Media

LB agar Described in Ausubel, F. M. et al. (eds.) “Current protocols inMolecular Biology”. John Wiley and Sons, 1995 LB-PG agar LB agarsupplemented with 0.5% Glucose and 0.05 M potassium phosphate, pH 7.0PS-1 10% sucrose, 4% soybean flour, 1% Na₃PO₄-12H₂O, 0.5% CaCO₃, and0.01% pluronic acid TE 10 mM Tris-HCl, pH 7.4 1 mM EDTA, pH 8.0 TEL 50mg/ml Lysozym in TE-buffer Thiocyanate 5 M guanidium thiocyanate 100 mMEDTA 0.6% w/v N-laurylsarcosine, sodium salt 60 g thiocyanate, 20 ml 0.5M EDTA, pH 8.0, 20 ml H₂O dissolves at 65° C. Cool down to roomtemperature (RT) and add 0.6 g N-laurylsarcosine. Add H₂O to 100 ml andfilter it through a 0.2 micron sterile filter. NH₄Ac 7.5 M CH₃COONH₄ TER1 microgram/ml Rnase A in TE-buffer CIA Chloroform/isoamyl alcohol 24:1Experimental Procedure

SEQ ID NO: 1 is the DNA sequence encoding a proform of the protease fromNocardiopsis alba DSM 15647. Nucleotides 502–1065 corresponds to themature peptide encoding part.

SEQ ID NO: 2 is the deduced amino acid sequence of SEQ ID NO: 1. Aminoacids −167 to −1 is the propeptide, and amino acids 1 to 188 the maturepeptide.

Cloning of SEQ ID NO: 1

The wild type was grown for 3 days before harvest in the followingmedium at 30° C.:

Trypticase  20 g Yeast extract   5 g Ferrochloride   6 mgMagnesiumsulfate  15 mg Distilled water ad 1000 mlpH adjusted to 9 by addition of sodium carbonate

Genomic DNA from Nocardiopsis alba DSM 15647 was isolated according tothe following procedure:

-   Harvest 1.5 ml culture and resuspend in 100 microliters TEL.    Incubate at 37° C. for 30 min.-   Add 500 microliters thiocynate buffer and leave at room temperature    for 10 min.-   Add 250 microliters NH₄Ac and leave at ice for 10 min.-   Add 500 microliters CIA and mix.-   Transfer to a microcentrifuge and spin for 10 min. at full speed.-   Transfer supernatant to a new Eppendorf tube and add 0.54 volume    cold isopropanol. Mix thoroughly.-   Spin and wash the DNA pellet with 70% EtOH.-   Resuspend the genomic DNA in 100 microliters TER.

The genomic DNA was used as template for PCR amplification using belowprimers SEQ ID NOs: 3 and 4. The PCR fragment was isolated on a 0.7%agarose gel.

Primers:

Primers: 1421: 5′-GTT CAT CGA TCG CAT CGG CTG CGA CCG GCC CCC TCC CCCAGT C-3′ (SEQ ID NO: 3) 1604: 5′-GCG GAT CCT ATC AGG TGC GCA GGG TCA GACC-3′ (SEQ ID NO: 4)

The digested and purified PCR fragment was ligated to the Cla I andBamHI digested plasmid pDG268NeoMCS-PramyQ/PrcryIII/cryIIIAstab/Sav(U.S. Pat. No. 5,955,310).

The ligation mixture was used for transformation into E. coli TOP10F′(Invitrogen BV, The Netherlands) and several colonies were selected forminiprep (QIAprep spin, QIAGEN GmbH, Germany). The purified plasmidswere checked for insert before transformation into a strain of Bacillussubtilis derived from B. subtilis DN 1885 with disrupted apr, npr andpel genes (Diderichsen et al (1990), J. Bacteriol., 172, 4315–4321). Thedisruption was performed essentially as described in “Bacillus subtilisand other Gram-Positive Bacteria,” American Society for Microbiology, p.618, eds. A. L. Sonenshein, J. A. Hoch and Richard Losick (1993).Transformed cells were plated on 1% skim milk LB-PG agar plates,supplemented with 6 micrograms/ml chloramphenicol. The plated cells wereincubated over night at 37° C. and protease containing colonies wereidentified by a surrounding clearing zone. Protease positive colonieswere selected and the coding sequence of the expressed enzyme from theexpression construct was confirmed by DNA sequence analysis.

Fermentation

The Bacillus subtilis host cell transformed as described above wasfermented on a rotary shaking table (250 r.p.m.) in 500 ml baffledErlenmeyer flasks containing 100 ml PS-1 medium supplemented with 6micrograms/ml chloramphenicol, at 37° C. for 16 hours and at 26° C. forextra 4 days.

Example 2 Purification and Characterization of the Protease fromNocardiopsis alba DSM 15647.

Protease Assays

-   1) pNA assay:-   pNA substrate: Suc-AAPF-pNA (Bachem L-1400).-   Temperature: Room temperature (25° C.)-   Assay buffers: 100 mM succinic acid, 100 mM HEPES, 100 mM CHES, 100    mM CABS, 1 mM CaCl₂, 150 mM KCl, 0.01% Triton X-100 adjusted to    pH-values 2.0, 2.5, 3.0, 3.5, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0,    11.0, and 12.0 with HCl or NaOH.

20 microliters protease (diluted in 0.01% Triton X-100) is mixed with100 μl assay buffer. The assay is started by adding 100 microliters pNAsubstrate (50 mg dissolved in 1.0 ml DMSO and further diluted 45× with0.01% Triton X-100). The increase in OD₄₀₅ is monitored as a measure ofthe protease activity.

2) Protazyme AK Assay:

-   Substrate: Protazyme AK tablet (cross-linked and dyed casein; from    Megazyme)-   Temperature: controlled (assay temperature).-   Assay buffers: 100 mM succinic acid, 100 mM HEPES, 100 mM CHES, 100    mM CABS, 1 mM CaCl₂, 150 mM KCl, 0.01% Triton X-100 adjusted to    pH-values 2.0, 2.5, 3.0, 3.5, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 and    11.0 with HCl or NaOH.

A Protazyme AK tablet is suspended in 2.0 ml 0.01% Triton X-100 bygentle stirring. 500 microliters of this suspension and 500 microlitersassay buffer are mixed in an Eppendorf tube and placed on ice. 20microliters protease sample (diluted in 0.01% Triton X-100) is added.The assay is initiated by transferring the Eppendorf tube to anEppendorf thermomixer, which is set to the assay temperature. The tubeis incubated for 15 minutes on the Eppendorf thermomixer at its highestshaking rate (1400 rpm). The incubation is stopped by transferring thetube back to the ice bath. Then the tube is centrifuged in an icecoldcentrifuge for a few minutes and 200 microliters supernatant istransferred to a microtiter plate. OD₆₅₀ is read as a measure ofprotease activity. A buffer blind is included in the assay (instead ofenzyme).

The protease fermentation described in Example 1 was centrifuged (20000×g, 20 min) and the supernatants were carefully decanted from theprecipitates. The combined supernatants were filtered through a SeitzEKS plate in order to remove the rest of the Nocardiopsis cells. The EKSfiltrate was transferred to 50 mM H₃BO₃, 5 mM succinic acid, 1 mM CaCl₂,pH 7 on a G25 sephadex column which resulted in a turbid solution. Theturbidity was removed by another filtration through a Seitz EKS plate.The clear filtrate was applied to a bacitracin silica columnequilibrated in the same buffer. After washing the column extensivelywith the equilibration buffer, the protease was step-eluted with 100 mMH₃BO₃, 10 mM succinic acid, 2 mM CaCl₂, 1 M NaCl, 25% isopropanol, pH 7.The bacitracin eluate was transferred to 50 mM H₃BO₃, 5 mM succinicacid, 1 mM CaCl₂, pH 7 on a G25 sephadex column and concentrated byultrafiltration to a minimal volume in an Amicon concentration cellequipped with a 5000Da cut-off membrane. The concentrated enzyme wasapplied to a Superdex 75 size-exclusion column equilibrated in 100 mMH₃BO₃, 10 mM succinic acid, 2 mM CaCl₂, 200 mM NaCl, pH 7 and the columnwas eluted with the same buffer. Fractions from the column were analysedfor protease activity (using the Protazyme AK assay at 37° C. and pH 9)and active fractions were further analysed by SDS-PAGE. Fractions, whereonly one band was seen on the coomassie stained SDS-PAGE gel, werepooled as the purified preparation and was used for furthercharacterization.

pH-activity, pH-stability, and Temperature-activity

The pNA assay was used for obtaining the pH-activity profile as well asthe pH-stability profile. For the pH-stability profile the protease wasdiluted 10× in the assay buffers and incubated for 2 hours at 37° C.After incubation the protease samples were transferred to the same pH—pH9, before assay for residual activity, by dilution in the pH 9 assaybuffer.

The Protazyme AK assay was used for obtaining the temperature-activityprofile at pH 9. The results are shown in Tables 1–3 below.

TABLE 1 pH-activity profile Protease derived Protase derived fromNocardiopsis from Nocardiopsis sp. pH alba DSM 15647 NRRL 18262 2 0.00 —3 0.00 0.00 4 0.01 0.02 5 0.06 0.07 6 0.18 0.21 7 0.37 0.44 8 0.69 0.679 0.99 0.88 10 1.00 1.00 11 0.95 0.93

TABLE 2 pH-stability profile Protease Protase derived from derived fromNocardiopsis alba Nocardiopsis sp. pH DSM 15647 NRRL 18262 2.0 1.04 0.782.5 1.05 1.00 3.0 1.00 1.03 3.5 1.00 0.98 4.0 0.93 0.99 5.0 1.00 1.026.0 1.00 1.00 7.0 1.00 1.01 8.0 1.03 0.98 9.0 1.02 0.99 10.0  0.96 0.9911.0  0.95 0.86 12.0  0.88 — 9.0 and after 1.00 1.00 2 hours at 5° C.

TABLE 3 Temperature activity profile Protease Protase derived fromderived from Temperature Nocardiopsis alba Nocardiopsis sp. (° C.) DSM15647 NRRL 18262 15 0.02 0.02 25 0.05 0.02 37 0.10 0.07 50 0.27 0.20 600.56 0.51 70 1.00 1.00 80 0.49 0.39 90 — —

The protease was found to be inhibited by Phenyl Methyl SulfonylFluoride. Its relative molecular weight as determined by SDS-PAGE wasM_(r)=19 kDa.

Differential Scanning Calorimetry (DSC)

DSC was used to determine temperature stability at pH 7.0 of theprotease derived from Nocardiopsis alba and from Nocardiopsis sp. NRRL18262. The purified proteases were dialysed over night at 4° C. against10 mM sodium phosphate, 50 mM sodium chloride, pH 7.0 and run on aVP-DSC instrument (Micro Cal) with a constant scan rate of 1.5° C./minfrom 20 to 100° C. Data-handling was performed using the MicroCal Originsoftware.

The resulting denaturation or melting temperatures, T_(m),'s were: Forthe protease of the invention derived from Nocardiopsis alba: 78.3° C.;for the protease derived from Nocardiopsis sp. NRRL 18262: 76.5° C.

Example 3 Specific Activity of the Protease from Nocardiopsis alba DSM15647

The purified protease preparation described in Example 2 was used fordetermination of the specific activity. The purity of the preparationwas above 95% when analysed by SDS-PAGE (determined as described inExample 2A in WO 01/58275). The protease sample was divided in two. Onepart was analysed for protein content (mg/ml) by amino acid analysis,the other part was analysed for protease activity.

Amino Acid Analysis (AAA)/(mg/ml)

The peptide bonds of the protease sample were subjected to acidhydrolysis, followed by separation and quantification of the releasedamino acids on a Biochrom 20 Plus Amino Acid Analyser, commerciallyavailable from Bie & Berntsen A/S, Sandbaekvej 5–7, DK-2610 Roedovre,Denmark, according to the manufacturer's instructions. For the acidhydrolysis, the protein sample was dried in a vacuum centrifuge,resolved in 18.5% (vol/vol) HCl+0.1% (vol/vol) phenol and incubated for16hr at 110° C. After incubation, the sample was again dried in thevacuum centrifuge, resolved in loading buffer (0.2 M Na-Citrate, pH 2.2)and loaded onto the Biochrom 20 Plus Amino Acid Analyser.

For the quantification, the hydrolysed sample was loaded onto a columnof the cation-exchange resin UltroPac no. 8, Sodium-form, which iscommercially available from Bie & Berntsen A/S, catalogue no.80-2104-15. Buffers of varying pH (pH 1 to pH 8) and ionic strength werepumped through the column according to the manufacturer's instructionsreferred to above, to separate the various amino acids. The columntemperature was accurately controlled, also according to themanufacturer's instructions (from 53° C. to 92° C. and back to 53° C.)in order to ensure the required separation. The column eluent was mixedwith ninhydrin reagent (Bie & Berntsen, catalogue no. 80-2038-07) andthe mixture passed through the high temperature reaction coil of theAmino Acid Analyser. In the reaction coil, ninhydrin reacted with theamino acids to form coloured compounds, the amount of which was directlyproportional to the quantity of amino acid present.

Protease Activity Assay (AU/ml)

Denatured haemoglobin (0.65% (w/w) in 6.7 mM KH₂PO₄/NaOH buffer, pH7.50) was degraded at 25° C. for 10 minutes by the protease, andundigested haemoglobin was precipitated with trichloroacetic acid (TCA)and removed by filtration. The TCA-soluble haemoglobin degradationproducts in the filtrate were determined with Folin & Ciocalteu's phenolreagent, which gives a blue colour with several amino acids. Theactivity unit (AU) was measured and defined by reference to an ALCALASE™standard. A detailed description of the assay, as well as a sample ofthe ALCALASE™ standard, is available on request from Novozymes A/S,Krogshoejvej 36, DK-2880 Bagsvaerd, Denmark (assay no.EB-SM-0349.02/01).

The specific activity was calculated as: Specific activity(AU/g)=(Activity (AU/ml)/AAA (mg/ml))×1000 (mg/g).

The specific activity of the protease derived from Nocardiopsis alba DSM15647 was 53.5 AU/g, as compared to the specific activity of theprotease derived from Nocardiopsis sp. NRRL 18262 of 38.3 AU/g.

Example 4 Protease L1a

Nocardiopsis dassonvillei subsp. dassonvillei DSM 43235 was grown in thewild type Trypticase medium, and genomic DNA isolated, as described inExample 1.

The coding region for the pro-mature protease L1a (nucleotides 88–1143of SEQ ID NO: 1) was amplified with the following primers 1424 and 1485on the genomic DNA:

-   Primer 1485 (SEQ ID NO: 7):    5′-gcttttagttcatcgatcgcatcggctgcgaccgtaccggccgagccag-3′-   Primer 1424 (SEQ ID NO: 8):    5′-ggagcggattgaacatgcgattactaaccggtcaccagggacagcc-3′

The L1 a polynucleotides were fused, by PCR, in frame to a heterologousDNA fragment encoding a Sav signal peptide (SEQ ID NO: 9).

A Bacillus subtilis strain designated Sav-L1a was constructed byincorporating the gene (including the signal peptide encoding part) byhomologous recombination on the Bacillus subtilis MB1053 host cellgenome (WO 03/95658). The gene was expressed under the control of atriple promoter system (as described in WO 99/43835), consisting of thepromoters from Bacillus licheniformis alpha-amylase gene (amyL),Bacillus amyloliquefaciens alpha-amylase gene (amyQ), and the Bacillusthuringiensis cryIIIA promoter including stabilizing sequence. The genecoding for Chloramphenicol acetyl-transferase was used as marker(described in eg. Diderichsen, B.; Poulsen, G. B.; Joergensen, S. T.; Auseful cloning vector for Bacillus subtilis. Plasmid 30:312 (1993)).

Chloramphenicol resistant transformants were checked for proteaseactivity and a transformant selected for sequence verification asdescribed in Example 1, following which it was fermented as alsodescribed in Example 1, but at 26° C. for 6 days.

The culture broth was centrifuged (20000×g, 20 min) and the supernatantswere carefully decanted from the precipitates. The combined supernatantswere filtered through a Seitz EKS plate in order to remove the rest ofthe Bacillus host cells. The EKS filtrate was transferred to 50 mMH₃BO₃, 5 mM succinic acid, 1 mM CaCl₂, pH 7 on a G25 sephadex column.Solid ammonium sulfate was added to the enzyme solution from the G25sephadex column to give a 1.6 M final (NH₄)₂SO₄ concentration in theenzyme solution. The enzyme solution was mixed gently with a magneticstirrer during the (NH₄)₂SO₄ addition and the stirring was continued for30 minutes after the addition to bring the system in equilibrium. Thenthe enzyme solution was applied to a Butyl Toyopearl column equilibratedin 100 mM H₃BO₃, 10 mM succinic acid, 2 mM CaCl₂, 1.6 M (NH₄)₂SO₄, pH 7.After washing the column extensively with the equilibration buffer, theprotease was eluted with a linear (NH₄)₂SO₄ gradient (1.6 to 0 M) in thesame buffer. Protease containing fractions were pooled and transferredto 20 mM HEPES, pH 8 on a G25 sephadex column and applied to a Qsepharose FF column equilibrated in the same buffer. After washing thecolumn extensively with the equilibration buffer, the protease waseluted with a linear NaCl gradient (0 to 0.5 M) in the same buffer.Fractions from the column were analysed for protease activity (using theSuc-MPF-pNA assay at pH 9) and active fractions were further analysed bySDS-PAGE. Fractions with only one band (as judged by a coomassie stainedSDS-PAGE gel) were pooled to provide the purified preparation which wasused for further characterization.

The L1a protease is an alpha-lytic protease like enzyme (peptidasefamily S1E, old notation S2A) which is found to be inhibited by PhenylMethyl Sulfonyl Fluoride (PMSF), and by the Streptomyces SubtilisinInhibitor (SSI). Its relative molecular weight as determined by SDS-PAGEis M_(r)=22 kDa.

The specific activity of the L1a protease was determined as described inExample 3 to 49.8 AU/g.

Example 5 Monogastric in vitro Results

The performance of the purified protease described in Example 2 wastested in an in vitro model simulating the digestion in monogastricanimals. In particular, the protease was tested for its ability toimprove solubilisation and digestion of maize/-SBM (maize/-soybean meal)proteins. The in vitro system consisted of 10 flasks in which maize/-SBMsubstrate was initially incubated with HCl/pepsin—simulating gastricdigestion—and subsequently with pancreatin—simulating intestinaldigestion. Five of the flasks were dosed with the protease at the startof the gastric phase whereas the remaining five flasks served as blanks.At the end of the intestinal incubation phase samples of in vitrodigesta were removed and analysed for solubilised and digested protein.

Outline of in vitro digestion procedure Simulated Components Timedigestion added pH Temperature course phase 10 g maize/-SBM substrate3.0 40° C. t = 0 min Mixing (6:4), 41 ml HCl (0.105 M) 5 ml HCl (0.105M)/pepsin 3.0 40° C. t = 30 min Gastric (3000 U/g substrate), 1 mldigestion protease (to provide 100 mg protease enzyme protein per kg ofsubstrate) 16 ml H₂O 3.0 40° C. t = 1.0 hour Gastric digestion 7 ml NaOH(0.39 M) 6.8 40° C. t = 1.5 hours Intestinal digestion 5 ml NaHCO₃ (1M)/ 6.8 40° C. t = 2.0 hours Intestinal pancreatin (8 mg/g diet)digestion Terminate incubation 7.0 40° C. t = 6.0 hoursConditions

-   Substrate: 4 g SBM, 6 g maize (premixed)-   pH: 3.0 stomach step/6.8–7.0 intestinal step-   HCl: 0.105 M for 1.5 hours (i.e. 30 min HCl-substrate premixing)-   pepsin: 3000 U/g diet for 1 hour-   pancreatin: 8 mg/g diet for 4 hours-   temperature: 40° C.-   Replicates: n    Solutions-   0.39 M NaOH-   0.105 M HCl-   0.105 M HCl containing 6000 U pepsin per 5 ml-   1 M NaHCO₃ containing 16 mg pancreatin per ml-   125 mM NaAc-buffer, pH 6.0

The amount of protease enzyme protein (EP) is calculated on the basis ofthe A₂₈₀ values and the amino acid sequences (amino acid compositions)using the principles outlined in S. C. Gill & P. H. von Hippel,Analytical Biochemistry 182, 319–326 (1989). The experimental procedurewas according to the above outline. pH was measured at time 1, 2.5, and5.5 hours. Incubations were terminated after 6 hours and samples of 30ml were removed and placed on ice before centrifugation (10000×g, 10min, 4° C.). Supernatants were removed and stored at −20° C.

All samples were analysed for content of solubilised and digestedprotein using gel filtration, and for degree of hydrolysis (DH) with theOPA method.

DH Determination by the OPA-method

The Degree of Hydrolysis of protein in different samples was determinedusing an semi-automated microtiter plate based colorimetric method(Nielsen, P. M.; Petersen, D.; Dambmann, C. Improved method fordetermining food protein degree of hydrolysis. J. Food Sci. 2001, 66,642–646). The OPA reagent was prepared as follows: 7.620 g di-Natetraborate decahydrate and 200 mg sodiumdodecyl sulphate (SDS) weredissolved in 150 ml deionized water. The reagents were completelydissolved before continuing. 160 mg o-phthal-dialdehyde 97% (OPA) wasdissolved in 4 ml ethanol. The OPA solution was transferredquantitatively to the above-mentioned solution by rinsing with deionizedwater. 176 mg dithiothreitol 99% (DTT) was added to the solution thatwas made up to 200 ml with deionized water. A serine standard (0.9516meqv/l) was prepared by solubilising 50 mg serine (Merck, Germany) in500 ml deionized water.

The sample solution was prepared by diluting each sample to anabsorbance (280 nm) of about 0.5. Generally, supernatants were diluted(100×) using an automated Tecan dilution station (Männedorf,Switzerland). All other spectrophotometer readings were performed at 340nm using deionized water as the control. 25 microliters of sample,standard and blind was dispensed into a microtiter plate. Themicro-titer plate was inserted into an iEMS MF reader (Labsystems,Finland) and 200 microliters of OPA reagent was automatically dispensed.Plates were shaken (2 min; 700 rpm) before measuring absorbance.Finally, the DH was calculated. Fivefold determination of all sampleswas carried out.

Estimation of Solubilised and Digested Protein

The content of solubilised protein in supernatants from in vitrodigested samples was estimated by quantifying crude protein (CP) usinggel filtration HPLC. Supernatants were thawed, filtered through 0.45micro-m polycarbonate filters and diluted (1:50, v/v) with H₂O . Dilutedsamples were chromatographed by HPLC using a Superdex Peptide PE(7.5×300 mm) gel filtration column (Global). The eluent used forisocratic elution was 50 mM sodium phosphate buffer (pH 7.0) containing150 mM NaCl. The total volume of eluent per run was 26 ml and the flowrate was 0.4 ml/min. Elution profiles were recorded at 214 nm and thetotal area under the profiles was determined by integration. To estimateprotein content from integrated areas, a calibration curve (R²=0.9993)was made from a dilution series of an in vitro digested referencemaize/-SBM sample with known total protein content. The proteindetermination in this reference sample was carried out using theKjeldahl method (determination of % nitrogen; A.O.A.C. (1984) OfficialMethods of Analysis 14th ed., Washington D.C.).

The content of digested protein was estimated by integrating thechromatogram area corresponding to peptides and amino acids having amolecular mass of 1500 Dalton or below (Savoie, L.; Gauthier, S. F.Dialysis Cell For The In-vitro Measurement Of Protein Digestibility. J.Food Sci. 1986, 51, 494–498; Babinszky,L.; Van, D. M. J. M.; Boer, H.;Den, H. L. A. An In-vitro Method for Prediction of The Digestible CrudeProtein Content in Pig Feeds. J. Sci. Food Agr. 1990, 50, 173–178;Boisen, S.; Eggum, B. O. Critical Evaluation of In-vitro Methods forEstimating Digestibility in-Simple-Stomach Animals. Nutrition ResearchReviews 1991, 4, 141–162). To determine the 1500 Dalton dividing line,the gel filtration column was calibrated using cytochrome C (Boehringer,Germany), aprotinin, gastrin 1, and substance P (Sigma Aldrich, USA), asmolecular mass standards.

The results shown in Tables 4 and 5 below indicate that the proteasesignificantly increased the level of digestible protein, as well as thedegree of hydrolysis, both relative to the blank.

TABLE 4 Degree of Hydrolysis (DH) Enzyme (dosage Relative to blank in mgEP/kg feed) n % DH % CV Blank 5 100.0 ^(a) 2.05 Protease of Example 2 5104.8 ^(b) 1.57Different letters within the same column indicate significantdifferences (1-way ANOVA, Tukey-Kramer test, P<0.05). SD=StandardDeviation. % CV=Coefficient of Variance=(SD/mean value)×100%

TABLE 5 Solubilised and digested crude protein Relative to blank EnzymeN % digestible CP CV % % soluble CP CV % Blank 5 100.0 ^(a) 2.3 100.0^(a) 2.4 Protease of 5 104.7 ^(b) 0.7 101.8 ^(a) 0.5 Example 2Different letters within the same column indicate significantdifferences (1-way ANOVA, Tukey-Kramer test, P<0.05). SD=StandardDeviation. % CV=Coefficient of Variance=(SD/mean value)×100%

Example 6 Animal Feed and Animal Feed Additives

An animal feed additive comprising the protease prepared as described inExample 2, the feed additive being in the form of a vitamins and mineralpremix, is composed as shown in Table 6 below. The vitamins and thecarotenoids are commercially available from DSM Nutritional Products.All amounts are in g/kg.

TABLE 6 Premix composition Vitamin A ROVIMIX A 500 4.00 Vitamin D3ROVIMIX D3 500 1.00 Vitamin E ROVIMIX E 50 Ads 8.00 Vitamin B2 ROVIMIXB2 80-SD 1.0 CAROPHYLL Yellow 10.0 Choline chloride 50%, min. 300.0Minerals Mn Oxide 60.0 Zn Oxide 12.0 Fe Sulphate monohydrate 20.0 CuOxide 2.0 Co Sulphate 0.2 Enzyme Protease of Example 2 10.0 Wheatmiddlings 571.8

The Premix of Table 6 is included in a diet for layers with acomposition as shown in Table 7 below. The amount of each ingredient isindicated in % (w/w). The concentration in the 5 diet of the L2aprotease is 100 mg protease enzyme protein per kg of the diet.

TABLE 7 Diet for layers Maize 55.00 Wheat 10.00 Oat 7.50 Soya 20.00Limestone 7.50 Premix of Table 6 1.00

1. An isolated polypeptide having protease activity, wherein the polypeptide consists of: a) a polypeptide having an amino acid sequence which has a degree of identity to amino acid 1–188 of SEQ ID NO: 2 or 1–192 of SEQ ID NO: 6 of at least 95%; b) a polypeptide which is encoded by a nucleic acid sequence which hybridizes under high stringency conditions with nucleotides 568–1143 of SEQ ID NO: 5; (c) a polypeptide which is encoded by a nucleic acid sequence which has a degree of identity to nucleotides 568–1143 of SEQ ID NO: 5 of at least 95%.
 2. The polypeptide of claim 1, which has an amino acid sequence which has a degree of identity to amino acids 1–192 of SEQ ID NO: 6 of at least 95%.
 3. The polypeptide of claim 1, which is encoded by a nucleic acid sequence which hybridizes under high stringency conditions with nucleotides 568–1143 of SEQ ID NO:
 5. 4. An animal feed additive comprising at least one polypeptide of claim 1; and (a) at least one fat soluble vitamin, and/or (b) at least one water soluble vitamin, and/or (a) at least one trace mineral.
 5. An animal feed composition having a crude protein content of 50 to 800 g/kg and comprising a polypeptide of claim
 1. 6. An animal feed composition having a crude protein content of 50 to 800 g/kg and a feed additive of claim
 4. 7. A composition comprising a polypeptide of claim 1, and at least one other enzyme selected from the group consisting of alpha-galactosidase, amylase, beta-glucanase, galactanase, phospholipase, phytase, protease, and/or xylanase.
 8. A detergent composition comprising a polypeptide of claim 1 and a surfactant.
 9. The polypeptide of claim 1, which has an amino acid sequence which has a degree of identity to amino acids 1–188 of SEQ ID NO: 2 of at least 95%.
 10. The polypeptide of claim 1, which is encoded by a nucleic acid sequence which hybridizes under high stringency conditions with nucleotides 502–1065 of SEQ ID NO:
 1. 11. A polypeptide having an amino acid sequence of 1–188 of SEQ ID NO:
 2. 12. A polypeptide having an ammo acid sequence of 1–192 of SEQ ID NO:
 6. 